Genomic mapping method by direct haplotyping using intron sequence analysis

The present invention is an improved genomic mapping method which is able to generate highly informative polymorphic sites throughout the genome. In addition to being highly polymorphic, the sites can be used to generate patterns that identify allelic and sub-allelic haplotypes associated with the region.

FIELD OF THE INVENTION 
The present invention directly identifies haplotypes of individuals by 
analysis of non-coding sequence variation. This invention has a wide range 
of applications to rapidly test polymorphisms at specific sites throughout 
the genome and to expedite positional cloning of unknown human genetic 
disease genes identified by unique phenotypes. 
BACKGROUND OF THE INVENTION 
The cloned disease genes have been used to define the types of mutations 
causing human genetic disease (S. H. Orkin, et al Ann. Rev. Genet 
18:131-171 (1984)), allowed the detection of abnormal genes prenatally 
(c.f. R. V. Lebo, et al Am. J. Hum. Genet 47:583-590 (1990)), and led to 
gene replacement therapy trials of those genes that can be introduced into 
the affected tissue (S. A. Rosenberg, et al Human Gene Therapy 1:73-92 
(1990)). On the way to the goal of ultimately sequencing the entire human 
genome, the Human Genome Project will generate considerable mapping data 
and isolate and map RFLPs sequence tagged sites (STS), and cDNAs (ESTs; 
expressed sequence tags; M. D. Adams, et al Sci. 252:1651-1656 (1991)). 
Currently the most common method of identifying polymorphic markers is by 
restriction enzyme analysis using numerous restriction endonucleases. This 
process is labor intensive. This invention proposes to generate 
considerably more informative sites rapidly to expedite genome mapping, to 
identify unknown disease genes, and to provide information for prenatal 
diagnosis of at-risk fetuses. 
HUMAN GENOME PROJECT 
The Human Genome Project is a logical extension of individual efforts to 
map human genes and identify genes important to understanding development, 
tissue-specific expression and human genetic disease. The difficulty is in 
the large size and vast amount of information: each haploid genome 
received from each human parent has 3.times.10.sup.9 basepairs of DNA. The 
initial long term goal of the Genome Project is to sequence each basepair 
from a normal person(s). Individual scientists with related projects are 
expanding the scope and cost of the project by including other related 
goals. The initial first step was to generate a map of polymorphic linked 
loci at about 10 centimorgans throughout the genome. Several 10 
centimorgan maps of individual chromosomes have been generated and a 
couple of chromosomes are nearing a 1 centimorgan map. In the meantime, 
the most dense polymorphic maps generated are in the regions of unknown 
disease genes with uniquely distinguishable phenotypes. Positional cloning 
projects have succeeded in identifying about a dozen unknown disease genes 
(see below) and have produced high density maps in the disease gene 
regions. The most useful markers in generating chromosome linkage maps are 
those polymorphic markers with many alleles that are informative in nearly 
every mating. These sites make the adjacent sites with fewer informative 
matings more informative. This invention proposes a means to develop 
considerably more informative polymorphic sites as anchor points for 
linkage studies. 
PRENATAL DIAGNOSIS OF CLONED DISEASE GENES 
About a dozen disease genes have been cloned based upon the known gene 
product like hemoglobin or clotting factor. Another dozen genes have been 
isolated by positional cloning. Initially prenatal diagnosis is offered 
based on the segregation of informative polymorphisms in the disease gene 
region. Standard RFLP analysis that identify enough informative 
polymorphisms to assure diagnoses in nearly each case can be more (R. V. 
Lebo, et al Am. J. Hum. Genet 47:583-590 (1990)) or less labor intensive. 
When a limited number of gene mutation result in most of the 
disease-causing sequence changes at any given locus, then specific probes 
for each mutation account for a significant portion of the disease 
alleles, polymorphic analysis of the abnormal haplotypes may be the only 
available option. Currently this is the case for cystic fibrosis with its 
many reported mutated alleles. However, use of a method that provides much 
more informative polymorphic sites which are screened than the present 
methods would expedite the ability to implement prenatal analysis. 
"Reverse genetics" or "positional cloning" of an unknown disease gene 
refers to the process of moving toward a genetic disease locus by ever 
closer flanking polymorphic markers that recombine ever less frequently 
until candidate genes can be isolated and sequenced in patients and normal 
subjects. The disease gene has been identified when all mutant alleles can 
be shown to have a disease-causing alteration (c.f. S. H. Orkin, et al 
Ann. Rev. Genet. 18:131-171 (1984)) and all normal alleles have normal 
sequences. The first part of the search sifts through many polymorphic 
markers throughout the genome until a polymorphism is found to give a LOD 
(log of the odds) score greater than 2. Then, more markers are tested 
until a LOD score of 3 is obtained, and the linkage is considered proven. 
This means the likelihood that the polymorphic site is linked to the gene 
is greater than 999/1000 (log.sub.10 1000=3; 10.sup.3 =1000). Ray White's 
laboratory finds that, as expected, about 1 putative linkage out of 1000 
tested with LOD scores greater than 3.0 is unlinked. 
The entire genome is estimated to include about 3000 centimorgans (1 
centimorgan=1% recombination) on the 22 pairs of autosomes and one pair of 
sex determining chromosomes. Ideally one would have available about 300 
evenly spaced very polymorphic sites at 10 cM intervals throughout the 
entire genome so that each search of the genome for linkage to a disease 
gene would reveal linkage between one informative polymorphic marker and 
the disease phenotype. Unfortunately only some chromosomes have well 
mapped polymorphic markers. Those markers are not evenly spaced on the 
chromosomes. Therefore, current genome searches for a linked polymorphic 
marker typically include about 1200 polymorphic probes that test about 85% 
of the total human genome. The more informative the chromosome site, the 
more useful. 
One goal of the human genome project is to develop evenly spaced, very 
polymorphic sites so that additional disease genes can be mapped readily 
using the fewest number of markers and patients possible. Isolating and 
mapping cDNAs from tissue-specific libraries will provide additional 
unique mapped chromosome sites as well as candidate genes for genetic 
diseases (M. D. Adams et al Sci. 252:1651-1656 (1991)). Once located, a 
search for informative polymorphic markers at that chromosome site is 
required so that segregation analysis between disease gene phenotypes 
and/or other polymorphic sites can add the locus to a genetic (linkage) 
map or test the site as a candidate gene. 
Another trend is that the predicted number of centimorgans based upon 
counting chiasma (recombinations) in early metaphase of male meiosis has 
underestimated the number of centimorgans in thoroughly studied 
chromosomes. For instance, chromosome 1 had been estimated to be 200 to 
300 centimorgans, but the genetic distance has now been demonstrated to be 
about 464 centimorgans to the most distal polymorphic sites tested. 
Therefore the number of polymorphic probes may have to be even greater 
than previously estimated to screen the entire genome for an unknown 
genetic disease phenotype. 
The number of affected patients and their families required prior to 
initiating a positional cloning project depends upon the mode of 
inheritance. Another factor is the probability that a polymorphic marker 
associated with the probe will only be informative in a portion of the 
matings. A good patient population to study for an autosomal recessive 
genetic disease is 20 families with two living children affected with the 
disease. This allows the investigator to determine the phase of the 
disease phenotype and polymorphic locus in all informative patients and 
the first affected child and to compare the rate of recombination in the 
second child. For autosomal dominant genetic diseases, a single large 
pedigree may have 10 informative meioses so that two such pedigrees will 
be sufficient for testing. This number of subjects can be expected to give 
a LOD score between +2.0 and +3.0 for an informative polymorphic marker 
with a minor allele frequency of 30% (the major allele frequency of a two 
allele system is then 70%). For all polymorphic sites that give LOD scores 
greater than +2.0, linkage is tested with more polymorphic probes in this 
chromosome region. For all probes with LOD scores less than -2.0 (chance 
of linkage is less than 1/100=10.sup.-2), linkage is considered to be 
excluded. 
When a genetic disease is mapped to a unique chromosome region with a LOD 
score of 3, other polymorphic markers in that chromosome region are tested 
and the results compared by multipoint linkage analysis on computer 
programs like LIPED developed by Jurg Ott. Multipoint analysis increases 
the likelihood that the linkage is correct by raising the LOD score 
(perhaps to 4.0 so that the likelihood of linkage is 9,999/10,000) or 
often quickly excludes this chromosome region by revealing double 
recombinants in smaller chromosome regions that lower the LOD score 
precipitously. 
It is noted that a positional cloning project should not be initiated 
unless the clinical status of each family member can be determined with a 
high degree of certainty. An exception to this rule occurred when the 
"depression" locus was reported to segregate with the short arm of 
chromosome 11 (chromosome 11p) in the Amish. In this instance, it was 
discovered that the phenotype analysis on which the linkage study was 
based was incorrect when two patients promptly developed severe depression 
shortly after the LOD scores with chromosome 11p polymorphisms were 
reported to be greater than 6 (the odds of linkage are greater and 
1,000,000/1,000,001). When the LOD scores were calculated based on 
correctly assigning these two phenotypes, the correlation disproved the 
linkage. This development has made population geneticists more skeptical 
than necessary for easily diagnosed diseases, but emphasizes the 
importance of a correct clinical diagnosis in each family member on which 
the linkage studies are based. 
When a disease gene has been mapped to a linked polymorphic probe, the next 
step is to isolate and test the segregation of many other polymorphic 
markers in the same chromosome region. Obtaining existing markers depends 
upon the clones and libraries available from previous studies of the same 
chromosome region or available cDNAs or other very polymorphic probes 
previously mapped to this chromosome region. The optimal strategy for 
generating new probes depends upon collaborating laboratories' resources 
and expertise. 
For instance, somatic rodent-human hybrid cells carrying the whole human 
chromosome to which the disease gene has been mapped can be irradiated. 
Then cell strains carrying only the chromosome region with the linked 
polymorphic probe can be isolated. Recombinant libraries are screened with 
human alu repetitive sequences to identify the human clones. This 
identifies many human clones because the alu sequence is repeated about 
300,000 times throughout the human genome. From these clones DNA 
polymorphic sites can be identified, and further linkage analysis done in 
the families. 
A second approach is to dissect a portion of the chromosome in the disease 
gene region, amplify the few collected chromosome segments with alu 
primers, and clone the amplified fragments. These fragments are then used 
to find polymorphisms in the disease gene region. The segregation of these 
polymorphic sites are tested in all affected pedigrees to further define 
the disease gene region. Dissected libraries have been made in about a 
dozen known genetic disease loci. 
Simultaneously other cloned genes mapped to the putative disease gene 
region can be tested for polymorphisms and the segregation of these genes 
tested in affected pedigrees. 
At this point a clinical prenatal genetic test might be offered that is 95% 
reliable and informative in at least 75% of the cases as defined in R. V. 
Lebo, et al Am. J. Hum. Genet. 47:583-590 (1990). Depending upon the 
frequency of the genetic disease, the patient population may be limited to 
merely mapping the location of the disease gene and finding closely linked 
genetic markers. Other than identifying the alteration in the gene this 
might be the case for perhaps 2,000 of the over 4,000 genetic diseases 
described (McKusick, Mendelian Inheritance in Man). 
The next goal of positional cloning is to delineate an unknown genetic 
disease locus between flanking markers that span no more than 1 megabase 
(Mg) or 1,000,000 basepairs of DNA. The continuing process of screening 
ever greater numbers of cloned DNA fragments in such small chromosome 
regions while minimizing the number of clones tested outside the region is 
the most productive. For instance, cosmid clones isolated from irradiated 
chromosome 17 hybrids and identifying clones on the long arm by 
hybridization to hybrids carrying only that chromosome region were used to 
saturate the neurofibromatosis-1 gene locus. Then again, a library of 
fragments from the cystic fibrosis chromosome region was made from a cell 
line carrying a nearby selectable gene that was retained in all hybrid 
cell strains. 
About a dozen disease genes have been identified by positional cloning 
including Duchenne muscular dystrophy (A. P. Monaco, et al, Nature 
323:646-650 (1986)) and chronic granulomatous disease (B. Royer-Pokora, et 
al Cold Sp. Harbor Symp. LI:169-176 (1986)) on the X chromosome, cystic 
fibrosis (J. M. Rommens, et al Sci. 245:1059-1065 (1989); J. R. Riordan, 
et al Sci. 245:1066-1073 (1989); B. S. Kerem, et al Sci. 245:1073-1080 
(1989)) on chromosome 7, and neurofibromatosis-1 on chromosome 17 (M. R. 
Wallace, et al Sci. 249:181-186 (1990)). The X chromosome genes and the 
neurofibromatosis-1 locus on chromosome 17 were identified more easily and 
quickly because chromosome rearrangements defined the disease gene region. 
More effort was required to delimit the cystic fibrosis gene, an autosomal 
recessive genetic disease to a megabase region with flanking markers by 
linkage analysis and disequilibrium. Then chromosome hopping with yeast 
artificial chromosome (YAC) libraries was used to isolate the remaining 
DNA segments prior to identifying the abnormal gene with YAC "hopping" and 
"linking" libraries (J. M. Rommens, et al Sci. 245:1059-1065 (1989). 
Then transcribed genes in the putative disease gene region may be 
identified by searching for conserved sequences between species, looking 
for CpG islands with restriction endonuclease cut sites, and a recently 
developed exon trapping protocol. YAC clones are partially digested and 
subcloned in cosmids. Then the cosmids are labeled, preannealed to total 
unlabeled human DNA to hybridize to the repetitive sequences, and then 
hybridized to Souther blots of DNAs from dog, mouse, cat, and cow 
(referred to as zoo blots). YAC clones that carry sequences that hybridize 
to each are considered to carry conserved genes. These cosmids are then 
used to screen a tissue specific cDNA library. These cosmids recognizing 
homologous cDNA clones are then hybridized to Northern blots of different 
tissues to determine whether the putative gene is expressed in the 
affected tissue. Such tissues can come from a human cadaver or a 
laboratory rat. 
A second approach to identify genes is to cut YAC clones with enzymes like 
BssHII, EagI and SacII that recognize and cut CpG islands 5' to many genes 
(C. A. Sargent et al EMBO 8:2305-2312 (1989)). The isolated YAC clones can 
be digested and separated using pulsed field gel electrophoresis to learn 
whether any YAC inserts have been cut. Then the ends of the cut fragments 
can be isolated by ligating to plasmid vectors, digesting with an 
restriction enzyme that does not cut the vector, and transforming bacteria 
that require the plasmid to grow, just as the NotI YAC linking libraries 
were constructed. Plasmids that grow are used to screen zoo blots for 
conserved sequences, Northern blots of tissue extracts, and cDNA 
libraries. 
A third approach, Exon Trapping, has been developed by Dr. Geoffrey Duyk, 
who used retroviral vectors to help characterize gene regions. YAC or 
cosmid cloned sequences are digested and shotgun cloned into retroviral 
vector pETV-SD carrying an exon trap cassette. This Exon Trap vector 
identifies functional splice acceptor sites encoded in cloned genomic DNA 
fragments. Since most genes undergo RNA splicing, such sites serve as 
identifiers for most genes. Pooled plasmid DNA from this shotgun cloning 
is transfected into an ecotropic retroviral packaging cell line. This cell 
line provides proteins required for vector propagation as a retrovirus. 
Retroviral DNA is transcribed in vivo and transcripts with functional 
splice sites may undergo splicing with loss of the marked intervening 
sequence in the cassette. Spliced and unspliced viral RNAs are packaged 
into virions, harvested from this culture, and used to infect COS cells. 
This second replication increases the splicing frequency. Virus isolated 
from this second culture is used to infect COS cells that constitutively 
produce SV40 antigen. Thus the shuttle vector is reverse transcribed and 
amplified as a circular DNA episome with an SV40 origin or replication in 
the vector. The .beta.-galactosidase indicator gene is excised by splicing 
and results in a white colony whereas colonies that are not spliced are 
usually blue. Splicing events are verified in white colonies by DNA 
sequencing primed from within the splice donor exon. These candidate exons 
are used to screen zoo blots, cDNA libraries, or Northern blots to 
potentially identify genes. 
DESCRIPTION OF THE PRIOR ART 
Marx, Science 247:1540-1542 (1990) reports on the challenge that 
geneticists face in understanding components of multi-cause diseases such 
as autoimmune diseases, high blood pressure, obesity, cancer and mental 
diseases including Alzheimer's disease, manic depression, and 
schizophrenia. 
Olson et al, Science 245:1434-1435 (1989) reports a suggestion for a 
physical mapping system using sequence-tagged sites to provide a common 
language for genomic mapping. 
A series of three articles Rommens et al, Science 245:1059-1065 (1989), 
Riordan et al, Science 245:1066-1072 (1989) and Kerem et al, Science 
245:1073-1079 (1989) report a new gene analysis method called "jumping" 
used to identify the location of the CF gene, the sequence of the CF gene, 
and the defect in the gene and its percentage in the disease population, 
respectively. 
DiLelia et al, The Lancet i:497-499 (1988) describes a screening method for 
detecting the two major alleles responsible for phenylketonuria in 
Caucasians of Northern European descent. The mutations, located at about 
the center of exon 12 and at the exon 12 junction with intervening 
sequence 12 are detected by PCR amplification of a 245 bp region of exon 
12 and flanking intervening sequences. The amplified sequence encompasses 
both mutations and is analyzed using probes specific for each of the 
alleles (without prior electrophoretic separation). 
Dicker et al, BioTechniques 7:830-837 (1989) and Mardis et al, 
BioTechniques 7:840-850 (1989) report on automated techniques for 
sequencing of DNA sequences, particularly PCR-generated sequences. 
Clark, Mol. Biol. Evol., 7(2):111-122 (1990) describes an algorithm which 
can be used in some cases to infer haplotypes from PCR-generated allele 
determinations or to infer haplotype frequencies of closely linked 
restriction site polymorphisms. 
Stephens et al., Am. J. Hum. Genet., 46:1149-1155 (1990) describe a method 
for determining haplotypes of multiply heterozygous individuals. The 
method, referred to as "single-molecule-dilution" or "SDM", relies on 
stochastic separation of single-stranded DNA molecules by sufficient 
dilution to reliably include only one molecule of DNA in each diluted 
sample. Upon obtaining a single strand of DNA, the PCR technique is used 
to analyze the haplotype of the molecule. 
Another method for direct identification of haplotypes is described by 
Boehnke et al, Am. J. Hum. Genet. 45:21-32 (1989). The method performs the 
analysis using haploid cells, specifically sperm cells. 
Cavalli-Sforza, Am. J. Hum. Genet. 46:649-651 (1990) proposes that a 
statistical sampling of the genome of numerous individuals at various 
genetic locations be made as part of the genome mapping project to provide 
information as to the degree of individual variation present in the 
genome. 
Each of the above-described references is incorporated herein by reference 
in its entirety. 
SUMMARY OF THE INVENTION 
The present mapping method utilizes direct determination of haplotypes 
through analysis of an individual's genomic DNA. The present mapping 
method provides a way to obtain information regarding the amount of 
polymorphism associated with any genetic region of interest and to 
identify individuals having different alleles and haplotypes for the 
genetic region. In addition, the method provides information as to the 
distance and direction of a gene of interest, particularly a disease gene, 
from a given genetic locus. This method is particularly useful for 
locating disease genes that are not associated with chromosomal 
rearrangements. 
The method also provides a rapid way to generate polymorphic markers 
throughout the genome, particularly in any genetic locus of interest. Not 
only can the markers be identified and screened more readily than 
classical RFLP sites, but the markers are much more informative than 
classical RFLP sites, which are either present or absent at any given 
location. 
The present invention is based on the finding that non-coding region 
sequences, particularly intron sequences, contain genetic variations that 
are characteristic of alleles of adjacent and remote, linked genetic loci 
on the chromosome. In particular, primer-defined, amplified DNA sequences 
that include a sufficient number of intron sequence nucleotides can be 
used to produce patterns which are characteristic of alleles and 
haplotypes associated with a genetic region of interest. The patterns can 
be produced by gel electrophoresis length differences in the amplified DNA 
sequences or can be RFLP fragment patterns produced by digestion of the 
amplified DNA sequences with one or more endonucleases. Alternatively, 
once sufficient sequence information has been obtained, 
allele/haplotype-specific amplification can be used to detect the presence 
of the selected allele/haplotype. 
The mapping method provides information about the degree of polymorphism of 
a genetic locus by determining the number of allelic and sub-allelic 
(haplotypic) patterns produced for the locus by analyzing the DNA of 
numerous individuals. The method can be used to screen individuals to 
explore individual variation associated with a genetic locus of interest. 
The method also provides information regarding disease-associated genetic 
loci that can be used to study the population genetics of a disease, 
particularly monogenic disease. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention is an improved mapping method which is based on the 
ability to identify haplotypes of individuals through analysis of 
non-coding region sequence variation patterns, particularly intron 
sequence variation patterns. The mapping method has two aspects. First, 
for any particular region of interest, the method provides information 
regarding the degree of polymorphism associated with the region and 
identifies those individuals with differing allelic and sub-allelic 
(haplotypic) sequences, enabling characterization of individual 
variability throughout a population. For a particular region of interest, 
such characterization avoids repetitive sequencing of individuals with the 
same genetic sequence. 
By analyzing haplotype restriction associated with a region of interest, 
one can determine the direction of and, ultimately, the location of a gene 
of interest. In addition, direct haplotyping facilitates locating a 
disease-associated gene of interest without the need to resort to linkage 
analysis based on family studies. Direct analysis of haplotypes of normals 
and of those affected by the disease can be performed to identify the 
locus associated with a disease. 
Second, the method provides a rapid way to generate and screen polymorphic 
markers throughout the genome. In particular, non-coding sequences in any 
region for which there is about 200 to 500 nt of sequence information, 
particularly at a genetic locus, can be rapidly amplified and analyzed, 
and thus provide a marker which can be economically screened. In addition, 
the markers are much more informative than classical RFLP sites, which are 
either present or absent at any given location. For every genetic locus, 
analysis of one or a few intron sequence markers can identify the 
alleles/haplotypes associated with the locus. For intergenic sequences, 
the degree of polymorphism associated with the region is even higher. 
The present invention is based on the discovery that amplification of 
primer-defined DNA sequences that include a sufficient number of 
non-coding sequence nucleotides, particularly intron sequence nucleotides, 
can be used to produce patterns which are characteristic of alleles and 
haplotypes associated with a genetic region of interest. The present 
method reads haplotypes as the direct output of the DNA typing analysis 
when a single, individual organism is tested. The method is described 
herein in terms of mapping the human genome. However, the method is 
generally applicable to all eukaryotes. The method is preferably used for 
mapping genomic DNA of plant and animal species. 
Definitions 
The term "allele", as used herein, means a genetic variation associated 
with a coding region; that is, an alternative form of the gene. Such 
variations include "silent" variations which do not result in the 
substitution of an amino acid in the encoded protein. 
The term "linkage", as used herein, refers to the degree to which regions 
of genomic DNA are inherited together. Regions on different chromosomes do 
not exhibit linkage and are inherited together 50% of the time. Adjacent 
genes that are always inherited together exhibit 100% linkage. 
The term "linkage disequilibrium", as used herein, refers to the 
co-occurrence of two alleles at linked loci such that the frequency of the 
co-occurrence of the alleles is greater than would be expected from the 
separate frequencies of occurrence of each allele. Alleles that co-occur 
with frequencies expected from their separate frequencies are said to be 
in "linkage equilibrium". 
As used herein, "haplotype" is a region of genomic DNA on a chromosome 
which is bounded by recombination sites such that genetic loci within a 
haplotypic region are usually inherited as a unit. However, occasionally, 
genetic rearrangements may occur within a haplotype. Thus, the term 
haplotype is an operational term that refers to the occurrence on a 
chromosome of linked loci. 
As used herein, the term "intron" refers to untranslated DNA sequences 
between exons. The 5' flanking region including the promoter and 3' 
flanking region associated with a gene are referred to as a gene locus. 
The term "intergenic sequence" is used to refer to the spacing sequences 
between genetic loci which are not associated with a coding region and are 
colloquially referred to as "junk". 
As used herein, the term "amplified DNA sequence" refers to DNA sequences 
which are copies of a portion of a DNA sequence and its complementary 
sequence, which copies correspond in nucleotide sequence to the original 
DNA sequence and its complementary sequence. 
The term "complement", as used herein, refers to a DNA sequence that is 
complementary to a specified DNA sequence. 
The term "primer site", as used herein, refers to the area of the target 
DNA to which a primer hybridizes. 
The term "primer pair", as used herein, means a set of primers including a 
5' upstream primer that hybridizes with the 5' end of the DNA sequence to 
be amplified and a 3', downstream primer that hybridizes with the 
complement of the 3' end of the sequence to be amplified. 
The term "exon-limited primers", as used herein, means a primer pair having 
primers located within or just outside of an exon in a conserved portion 
of the intron, which primers amplify a DNA sequence which includes an exon 
or a portion thereof and not more than a small, para-exonic region of the 
adjacent intron(s). 
The term "intron-spanning primers", as used herein, means a primer pair 
that amplifies at least a portion of one intron, which amplified intron 
region includes sequences which are not conserved. The intron-spanning 
primers can be located in conserved regions of the introns or in adjacent, 
upstream and/or downstream exon sequences. 
The term "genetic locus", as used herein, means the region of the genomic 
DNA that includes the gene that encodes a protein including any upstream 
or downstream transcribed noncoding regions and associated regulatory 
regions. Therefore, an HLA locus is the region of the genomic DNA that 
includes the gene that encodes an HLA gene product. 
As used herein, the term "adjacent locus" refers to either (1) the locus in 
which a DNA sequence is located or (2) the nearest upstream or downstream 
genetic locus for intron DNA sequences not associated with a genetic 
locus. 
As used herein, the term "remote locus" refers to either (1) a locus which 
is upstream or downstream from the locus in which a DNA sequence is 
located or (2) for intron sequences not associated with a genetic locus, a 
locus which is upstream or downstream from the nearest upstream or 
downstream genetic locus to the intron sequence. 
The term "locus-specific primer", as used herein, means a primer that 
specifically hybridizes with a portion of the stated gene locus or its 
complementary strand and does not hybridize with other DNA sequences under 
the conditions used in the amplification method. A locus-specific primer 
pair defines an amplified DNA sequence that is present in a plurality of 
alleles of a genetic locus or all alleles of the locus. The locus-specific 
primer pair contains one locus-specific primer. The other primer of the 
pair can be common to a multiplicity of genetic loci or can also be a 
locus-specific primer. 
The term "sequence-specific primer" (SSP), as used herein, means a primer 
that specifically hybridizes with a sequence polymorphism present in one 
or more alleles of a genetic locus or their complementary strands but not 
present in all the alleles of the locus. The SSP does not hybridize with 
alleles of the genetic locus that do not contain the sequence polymorphism 
under the conditions used in the amplification method. A sequence-specific 
primer pair defines an amplified DNA sequence that is present in a number 
of alleles of a genetic locus but not in all alleles of the locus. The 
sequence-specific primer pair contains one sequence-specific primer. The 
other primer of the pair can be common to a multiplicity of primer pairs 
for the genetic locus or can also be a specific for the same group of 
alleles as the sequence-specific primer. 
The term "allele-specific primer" (ASP), as used herein, means a primer 
that specifically hybridizes with a sequence polymorphism present in one 
allele of a genetic locus or its complementary strand and not present in 
other alleles of the locus. The ASP does not hybridize with other alleles 
of the genetic locus under the conditions used in the amplification 
method. An allele-specific primer pair defines an amplified DNA sequence 
that is present in one allele of a genetic locus and is not present in 
other alleles of the locus. The allele-specific primer pair contains at 
least one allele-specific primer. The other primer can be common to a 
plurality of alleles. 
The term "haplotype-specific primer" (HSP), as used herein, means a primer 
that specifically hybridizes with a sequence polymorphism present in one 
haplotype associated with a genetic locus and one or more adjacent loci or 
its complementary strand and not present in other haplotypes associated 
with the locus. The HSP does not hybridize with other haplotypes of the 
genetic locus under the conditions used in the amplification method. A 
haplotype-specific primer pair defines an amplified DNA sequence that is 
present in one haplotype associated with a genetic locus and is not 
present in other haplotypes associated with the locus. The 
haplotype-specific primer pair contains at least one haplotype-specific 
primer. The other primer can be common to a plurality of haplotypes 
associated with the genetic locus and its adjacent loci or can also be a 
haplotype-specific primer. 
As used herein, the terms "endonuclease" and "restriction endonuclease" 
refer to an enzyme that cuts double-stranded DNA having a particular 
nucleotide sequence. The specificities of numerous endonucleases are well 
known and can be found in a variety of publications, e.g. Molecular 
Cloning: A Laboratory Manual by Maniatis et al, Cold Spring Harbor 
Laboratory 1982. That manual is incorporated herein by reference in its 
entirety. 
The term "restriction fragment length polymorphism" (or RFLP), as used 
herein, refers to differences in DNA nucleotide sequences that produce 
fragments of different lengths when cleaved by a restriction endonuclease. 
The term "HLA DNA", as used herein, means DNA that includes the genes that 
encode HLA antigens. HLA DNA is found in all nucleated human cells. 
Analysis of Non-coding Sequence Variation 
Studies of non-coding sequence variation at loci of the HLA gene complex 
have revealed a stellar array of polymorphic variability that can be used 
to discern the evolution of the loci of this complex and study linkage 
disequilibrium between closely linked genetic disease loci and unique HLA 
haplotypes. Most gene loci are more conserved than those of the HLA 
complex in which polymorphic variability is maximized. This is because 
conservation of most gene loci is necessary for survival. Thus the HLA 
region can serve as a paradigm for polymorphism throughout the genome for 
approaches to analysis of allelic variation within each gene locus. 
Linkage disequilibrium reflects the cosegregation of very close chromosome 
regions through many generations. Linkage disequilibrium is the opposite 
extreme of linkage equilibrium which reflects the random segregation of 
genes on difference chromosomes or in distant chromosome regions. The 
considerable allelic variability at each locus can be used to assess the 
segregation of polymorphisms at neighboring gene loci for the generation 
of high resolution linkage maps by detecting significant linkage 
disequilibrium between alleles of loci in linked regions extending over 
one or more megabases. 
The prior art describes numerous instances where polymorphic loci are used 
to study the frequency of cosegregation of other linked polymorphic loci 
or genes in human pedigrees as described in the background section. In 
every instance where polymorphisms were used, analysis of non-coding 
sequence variation of this invention can be applied. The applicability in 
the HLA region with its highly variable alleles demonstrates that other 
non-coding gene sequences will be informative and will increase the power 
of each method of analysis proportionally with the increase in the number 
of informative matings at each locus. 
For instance, few RFLPs have more than 3 alleles identified by any 
restriction enzyme. Those RFLPs with 6 or more alleles are informative in 
almost every mating so that these loci serve as anchor points along a 
chromosome for other restriction enzyme results. Each anchor point 
increases the information derived from the flanking polymorphic alleles 
because these less informative locations cannot be compared with each 
other as often as with the anchor point. The marker sites of this 
invention will provide more anchor points that can be tested more rapidly 
for less effort. 
Anchor points can be derived most easily from chromosomally sublocalized 
cDNA clones that by definition represent exons with the intervening 
sequences spliced out. Primers can be synthesized in different regions of 
the cDNA and used to amplify non- coding sequences to test for allelic 
variability in the different introns. Introns less than 2 kb are most 
easily amplified. When reverse genetic projects isolate putative genes by 
screening zoo blots, cDNA libraries, or by cloning restriction sites, the 
gene sequences can be analyzed and tested for polymorphisms using the 
present method. 
A similar or greater level of variability is found in intergenic regions 
because less selective pressure is maintained in these regions than for 
active genes. Therefore those sequences can also be used for the location 
of the markers of this invention. 
More specifically, using the methods and reagents of this invention, two 
types of non-coding sequence variation associated with genetic loci and 
intergenic sequences have been found. The first is allele-associated 
variation. That is, the non-coding sequence variation pattern associates 
with the allele type at an adjacent locus. The second type of variation is 
suballelic variation associated with alleles of remote linked loci 
(haplotypes). That is, sequence variations are present in a sub-population 
of individuals with the same allele. This sub-allelic variation is 
associated with alleles at remote loci (haplotypes). Such haplotypic 
differences may occur between sequences characterized as having the same 
haplotype. Such individual-limited or individual-specific variation is not 
common. 
Furthermore, an amplified DNA sequence that contains sufficient non-coding 
sequences, preferably intron sequences, will vary depending on the 
allele/haplotype-associated sequence present in the sample DNA. That is, 
the non-coding sequence, particularly the introns, contain genetic 
variations (e.g.; changes in the number or location of restriction sites 
or gel electrophoresis length polymorphisms due to insertions, deletions 
and/or sequence substitutions that effect electrophoretic mobility) which 
are associated with alleles at adjacent and remote loci. 
These non-coding sequence variation patterns can be generated anywhere 
there is about 200 to 500 bp of sequence information. In particular, the 
sites can be generated within cDNA gene sequences, within STS sites and 
within known classical RFLP sites. Of these locations, most preferred is 
the generation of intron variation sites within genes. The number of 
cloned genes stood at 945 in 1989, so there are numerous loci that can be 
used. In addition, when a region of the genome is of particular interest, 
there are techniques for identifying additional genes within the region. 
For example, in cases where a genetic disease locus is mapped to a unique 
chromosomal region with a LOD score of 3 or more (a region of about one 
megabase of DNA containing about 20 genes), any of the prior art 
techniques; e.g. exon trapping and zoo blot analysis, can be used in the 
region to identify additional genes within the region. Once additional 
genetic loci in the region are identified, other intron variation markers 
in the newly identified loci can be developed as described hereinafter. 
As stated previously, these polymorphic variation patterns can be readily 
identified for any genetic locus. Furthermore, the patterns are readily 
produced and analyzed for rapid screening. In addition, the patterns are 
highly informative so that the screening provides much more information 
than classical RFLP sites. This combination makes intron variation 
patterns ideal markers for expeditious mapping of disease-associated gene 
loci. 
In particular, one of the goals of the genome mapping project is to produce 
dense, evenly spaced markers throughout the genome. As stated previously, 
the usefulness of a marker is directly related to the degree of 
polymorphism of the marker. 
The intron variation patterns generated by the present method can be used 
to facilitate this process. Specifically, amplified intron sequences can 
generate a physical and genetic map that is more dense and more 
informative for genome searches than prior art maps using less informative 
markers. The intron-containing, amplified DNA sequences of this invention 
from unique gene regions provide easily screened and very informative 
markers that directly provide allele and haplotype information about the 
locus. In addition to being easily screened and highly informative, the 
intron variation patterns can be generated and scored faster and less 
expensively than classical RFLP patterns. This is of particular value in 
providing the most information in the shortest time for the least cost. In 
light of the likelihood that the genome is larger than presently 
estimated, and of the large costs and limited funding for the genome 
project, use of a method that provides more information in a short period 
of time is particularly advantageous. In addition, it is anticipated that 
the genes will be more evenly distributed along the length of a chromosome 
than empirically determined RFLP sites. 
It is estimated that there will be 50,000 genes throughout the human 
genome. Concentrating the initial activity on mapping and studying gene 
sequences, rather than intergenic sequences reveals many more candidate 
disease genes to facilitate matching genes to genetic diseases. Since 
genes are estimated to represent about two percent of the genome, this is 
an effective initial approach that identifies genes as the part of 
obtaining the initial information to produce polymorphic sites. The 
polymorphic marker sites of this invention can be produced in any of the 
known genetic loci. 
In addition to identifying sequence polymorphism patterns in a gene, any 
other region for which there are at least 200, preferably at least 500 bp 
of sequence information can also be used as a marker site to produce 
amplified DNA sequences that produce patterns that identify 
alleles/haplotypes associated with the region. As stated previously, when 
locating polymorphic pattern sites, either genetic locus, STS, or RFLP 
sites can be used to provide markers. The non-coding polymorphic variation 
patterns of this invention are at least as effective as screening for CA 
and GA repeats throughout the genome at STS sites in terms of the limited 
cost and time required for screening in comparison to classical RFLP 
analyses. In addition, polymorphic variation patterns of this invention 
can be substituted for RFLP sites in any mapping technique where isolated 
cDNAs have been mapped to the same chromosome region. In addition to 
providing more informative polymorphic markers for use in any of the prior 
art mapping and linkage analysis methods, the haplotypic patterns for a 
region can then be used to generate a haplocontig map, as described below. 
Once the haplotypes for a region are determined, the haplotypes for a 
distant region (about 0.01 to 2 million basepairs Mbp! away) from the 
first locus are then analyzed in the same manner. The next region to be 
analyzed is preferably sufficiently close to provide some haplotypic 
patterns characterized by PDLP or RFLP patterns which are shared with the 
previous locus. That is, there will be patterns for some of the same 
haplotypes at an adjacent locus. By analyzing the haplotypic patterns at a 
given location, the location of genetic loci and of haplotypic regions can 
be identified. This identification can localize the borders of linked loci 
and of the haplotypic regions. In this way, contiguous overlapping 
haplotypic regions (haplocontigs) can be analyzed to form a map. 
Analysis of the patterns of intron variation for a particular group of 
individuals can identify both the alleles and subtypic or sub-allelic 
groups (haplotypes) at that locus present in members of the group. By 
analyzing a groups of individuals representing the greatest ethnic 
diversity among humans and greatest breed and species variety in plants 
and animals, patterns characteristic of the most common alleles/haplotypes 
of the locus can be identified. Additional allelic and haplotypic patterns 
can be identified by screening larger populations. In this way the degree 
of polymorphism in alleles/haplotypes associated with any locus of 
interest can be determined without the need to perform repetitive 
sequencing of numerous individuals. 
Rather than generating a map, the haplotypic pattern can be used to 
identify the direction of any gene of interest. Such genes include 
disease-associated genes and, in plants and animals, commercially 
desireable trait loci. For example, analysis of haplotypic patterns of 
patients with a disease, particularly a monogenic disease, produces 
patterns characteristic of the alleles/haplotypes of those patients at any 
locus. Then the direction of the disease associated gene from the locus 
can be determined. Specifically, by analyzing the degree of polymorphism 
associated with a particular trait at a given locus, the telomeric or 
centromeric direction of the location of the locus (locus-directional 
haplotyping) associated with the trait can be identified. That is, as the 
degree of polymorphism (the number of haplotypes) associated with the loci 
of individuals having the trait decreases, the locus is closer to the gene 
of interest. The locus associated with the trait will exhibit the highest 
degree of haplotype heterogeneity restriction. Conversely, as one moves 
further from the locus associated with a trait, the number of haplotypes 
of those with the trait increases until the gene associated with the trait 
is so far from the locus that those with the trait will have the same 
haplotypes as those without the trait for that locus. 
In some cases, the next analyzed locus will not share patterns with the 
first-tested locus. When the haplotypic patterns at the next locus are not 
consistent with the previous locus patterns, either recombination has 
resulted in generation of a new haplotype involving alleles on both sides 
of the site of recombination and/or the border of the haplotypic region 
has been crossed. That is, the second locus is on the other side of a site 
of a recombination. This phenomenon is found most frequently around 
recombination hot spots. Such loci may be separated by the presence of 
another haplotypic region intermediate between two tested loci. A DNA 
sequence intermediate between the evaluated loci can then be analyzed to 
determine the borders of the haplotypic region or to locate each of the 
genetic loci associated with the haplotype. 
Once the locations of the haplotypic region are established, the genetic 
loci within each border and, preferably, one central locus can be 
evaluated to determine the number of alleles at each locus and at the 
adjacent loci. This evaluation can determine the genetic variation 
patterns produced by the common alleles for each locus of a haplotypic 
region. 
By evaluating the adjacent region of the chromosome in the same manner, the 
mapping method determines overlapping haplotypic regions (haplocontigs) in 
a selected region of a chromosome and can be used to establish the 
haplotypic regions throughout the genome. 
In another aspect, the mapping method provides information regarding the 
degree of polymorphism associated with a genomic region of interest. By 
analyzing DNA from numerous individuals, the number of alleles and of 
haplotypes associated with a region of interest can be determined. PDLP 
and RFLP patterns produced in the analyses are more numerous and more 
closely linked to the genetic locus than RFLP sites located by classical 
methods, since the present method can effectively utilize all of the RFLP 
sites in the amplified DNA sequences. Allele/haplotype-specific 
amplification is the most preferred analysis method to quickly screen a 
locus. However, the method requires more sequence information prior to its 
implementation than PDLP or RFLP techniques. 
Minimum sequencing enables sequence comparisons between genetically 
disparate individuals. In this way one can identify non-homologous regions 
and make rational selection of sites for restriction analysis or ASP HSP 
amplification. Then, additional individuals associated with particular 
patterns of variability in a population can be identified prior to 
sequencing. This analysis eliminates duplicative sequencing of individuals 
with the same haplotype. Prior to sequencing a genetic locus of interest, 
the allelic and sub-allelic haplotypic patterns associated with the locus 
are determined. Only one individual with a particular sub-allelic 
haplotypic pattern need be sequenced. However, preferably two or three 
individuals are sequenced to confirm that the selected sequence is 
representative of the haplotype rather than represents an individual 
variation. In this way, all the common alleles for a haplotypic region 
together with characteristic sub-allelic (haplotypic) groups can be 
identified and sequenced. Furthermore, individuals with relatively rare 
haplotypes, such as those associated with a rare genetic disease, can be 
recognized by direct evaluation of haplotypic patterns in individuals with 
the disease. 
In particular, disease-associated alleles and haplotypes are revealed by 
restriction of the allele/haplotype heterogeneity in patients with a 
particular disease. That is, patients with a particular disease will have 
only a few of the alleles/haplotypes which are present in the general 
population. For example, of the greater than 100 HLA haplotypes comprising 
alleles of the DRB/DQA/DQB loci, only about 30 of the haplotypes are 
associated with diabetes. Of those 30 haplotypes, only five or six 
haplotypes are associated with early onset disease in chinese and 
Caucasians. 
Analysis of the individual variability for all genetic loci can identify 
previously unrecognized loci associated with monogenic disorders. The 
locus will be so tightly linked to the disease-associated gene that no 
recombination between the locus and the disease- associated gene will be 
observed. Further, the haplotypes associated with the disease will be 
restricted. That is, only a limited number of the haplotypes present in 
the normal population are also present in those patients with the disease. 
Identification and analysis of genes associated with multigenic disorders 
can be performed in the same manner. Specifically, haplotypes associated 
with the disease will also be restricted in patients with the disease at 
each locus associated with the disease. 
Location of Amplified DNA Sequence 
Amplified DNA sequences containing from about 200 to 500 nt corresponding 
to intron sequences can be used to characterize the allele associated with 
the intervening sequence, particularly if the intervening sequence is 
adjacent to a variable exon of the locus. Furthermore, amplified DNA 
sequences containing from about 200 to 2,000 nt, preferably 400 to 500 nt, 
corresponding to non-coding sequences associated with genetic loci, 
preferably intervening sequences, reflect patterns subtypic of alleles 
which are characteristic of the haplotype. Therefore, amplified DNA 
sequences corresponding to non-coding sequences associated with genes, 
preferably introns, are produced to analyze the adjacent locus and remote 
locus alleles. 
The amplified DNA sequence will necessarily be located in a region where 
there is sufficient sequence information to select primer sites. For 
disease-associated genes, frequently sequences for the region surrounding 
an RFLP site that correlates with the disease is known. Alternatively, the 
gene of interest may be known to be located near another genetic locus. In 
addition, if no further information is available, the STS sites 
characterized for the genome mapping project include sequence information 
for about 400 to 500 nucleotides and can be used. 
When genomic DNA sequences are available, primers are located to produce an 
amplified DNA sequence corresponding to an intervening sequence. If the 
location of the variable exon(s) for a locus is known, the amplified DNA 
sequence is preferably located in an intron adjacent to the variable exon. 
More preferably, the amplified DNA sequence will span the variable exon 
and include a portion, preferably the majority, most preferably all, of 
both adjacent introns. 
When only cDNA sequences are available and intron locations within the 
sequence are not identified, primers are selected at intervals of about 
200 nt and used to amplify genomic DNA. If the amplified sequence contains 
about 200 nt, the location of the first primer is moved about 200 nt to 
one side of the second primer location and the amplification is repeated 
until either (1) an amplified DNA sequence that is larger than expected is 
produced or (2) no amplified DNA sequence is produced, indicating the 
presence of an intervening sequence that is too large for the 
amplification method. In either case, the location of an intron sequence 
has been determined. 
When the primers span an intron and produce an amplified DNA sequence, the 
primers can be used on the DNA of numerous individuals to begin the 
analysis process. When no amplified DNA sequence is produced, the intron 
sequence defined by the primers may be too large. Either another intron 
sequence can be located, as described previously or anchored, one-sided 
amplification can be performed to produce a sequence corresponding to a 
portion of the intron. Those DNA sequences, or portions thereof, can be 
sequenced to locate a second primer site within the intron sequence to 
define an amplified DNA sequence for analysis. 
The amplified DNA sequence is defined (by selection of the location of the 
primer sites) to contain mostly intron, rather than exon sequences, when 
the sequences of introns and exons (or the exon sequences and locations of 
exon-intron junctions) in the region to be amplified are known. Primer 
selection and preparation methods, as well as DNA amplification methods, 
are well known and are described in detail hereinafter. 
Once an amplified DNA sequence containing intron sequences is produced, the 
primers are used to produce the corresponding amplified DNA sequences from 
a number of individuals. The sizes of the sequences and the fragment 
patterns using several restriction endonucleases can be examined to select 
an analytical method that demonstrates allelic and sub-allelic genetic 
diversity associated with the locus. Selection of endonucleases and 
production and analysis of RFLP patterns is well known and is described in 
detail hereinafter. 
Once a primer pair or primer pair/endonuclease combination is selected, the 
DNA of about 100 individuals would be amplified and the lengths of the 
sequences (fragments) would be determined. The lengths of the sequences 
will fall into patterns related to the allele(s)/haplotype(s) of the 
individuals for that amplified sequence. For each chromosomal region, 
amplified sequences could be produced at various points along the region 
to determine each allele/haplotype associated with the region. An 
exemplary identification of the alleles/haplotypes found in one haplotypic 
region of the human genome is the identification of 35 of the known HLA 
haplotypes of the DRB/DQA/DQB loci. That analysis is described in detail 
in the examples. As will be readily apparent from the example, new 
patterns associated with rare haplotypes or haplotypes associated with 
other population groups can be readily identified by performing the 
analysis. 
In a search for a gene associated with a genetic disease, there may be 30 
to 40 haplotypes associated with the DNA region containing the gene. The 
sequence variation for those with the disease would be restricted to a 
relatively small number (3 to 8) of disease-associated haplotypes that 
would account for a substantial percentage (about 70%) of the disease 
population. Those haplotypes produce different patterns upon amplification 
and analysis. The DNA from a relatively small number of individuals with 
the disease, of the order of 20 to 100, can be quickly amplified, digested 
and analyzed. The patterns will fall into 3 to 8 groups, depending on the 
number of allelic variations of the locus. Only one representative sample 
of DNA from each allelic group need be sequenced. Preferably, two or three 
samples are sequenced to confirm the samples are representative. In this 
way, one can determine those individuals who have different 
haplotype-associated alleles prior to sequencing and avoid repeated 
sequencing of the most prevalent haplotypes. 
For example, cystic fibrosis (CF) is an autosomal recessive disease, 
requiring the presence of a mutant gene on each chromosome. CF is the most 
common genetic disease in Caucasians, occurring once in 2,000 live births. 
It is estimated that one in forty Caucasians are carriers for the disease. 
Recently a specific deletion of three consecutive basepairs in the open 
reading frame of the putative CF gene leading to the loss of a 
phenylalanine residue at position 508 of the predicted 1480 amino acid 
polypeptide was reported Kerem et al, Science 245:1073-1080 (1989)!. 
Based on haplotype analysis, the deletion may account for most CF 
mutations in Northern European populations (about 68%). A second mutation 
is reportedly prevalent in some Southern European populations. Additional 
data indicate that several other mutations may cause the disease. 
Studies of haplotypes of parents of CF patients (who necessarily have one 
normal and one disease-associated haplotype) indicated that there are at 
least 178 haplotypes associated with the CF locus. Of those haplotypes, 90 
are associated only with the disease; 78 are found only in normals; and 10 
are associated with both the disease and with normals (Kerem et al, 
supra). The disease apparently is caused by several different mutations, 
some in very low frequency in the population. As demonstrated by the 
haplotype information, there are more haplotypes associated with the locus 
than there are mutant alleles responsible for the disease. 
The present method directly determines haplotypes associated with the locus 
and can detect haplotypes among the 178 currently recognized haplotypes 
associated with the disease-associated genetic locus. Additional 
haplotypes associated with the disease are readily determined through the 
rapid analysis of DNA of numerous CF patients by the methods of this 
invention. Furthermore, any mutations which may be associated with 
noncoding regulatory regions can also be detected by the method and will 
be identified by the screening process. 
Rather than attempting to determine each defect in a coding region that 
causes the disease, the present method amplifies intron sequences 
associated with the locus to identify allelic and sub-allelic patterns. 
New PDLP and RFLP patterns produced by intron sequences indicate the 
presence of a previously unrecognized haplotype. 
A detailed description of selection of primers, amplification methods, and 
analysis of DNA sequences is provided below. Those techniques can be used 
initially to determine the patterns produced by common alleles/haplotypes 
associated with the locus. Once the common patterns are established, the 
patterns can be refined so that additional haplotypes associated with a 
locus can be distinguished. This additional characterization allows new 
haplotypes to be more readily identified. This complete characterization 
is particularly valuable for loci associated with inherited diseases and 
for other medically important loci such as the HLA loci. However, for some 
loci, analysis of hundreds of individuals will demonstrate that there are 
only one or two haplotypes associated with the locus. Such small numbers 
indicate that a limited number of haplotypes are associated with the 
region. Analysis of another 100 individuals may provide an additional one 
or two haplotypes. DNA from numerous other individuals needs to be 
analyzed to detect haplotypes with a low frequency. Further 
characterization may not be desired for all such loci. 
There are three major types of genetic variations that can be detected 
within an amplified DNA sequence and used to identify allelic and 
sub-allelic groups. Those variations, in order of ease of detection, are 
(1) a change in the length of the sequence, (2) a change in the presence 
or location of at least one restriction site and (3) the substitution of 
one or a few nucleotides that does not result in a change in a restriction 
site. Other variations within the amplified DNA sequence are also 
detectable. Alternatively, once sufficient sequence information about the 
locus has been developed, allele/haplotype-specific amplification can be 
performed to analyze DNA of numerous additional individuals for that 
locus. Allele/haplotype specific amplification is based on selection of 
primer sites that are characteristic of the allele/haplotype. 
There are four types of techniques which can be used to detect the 
variations. The first is sequencing the amplified DNA sequence. Sequencing 
is the most time consuming and also the most revealing analytical method, 
since it detects any type of genetic variation in the amplified sequence. 
The second analytical method uses allele-specific oligonucleotides or 
sequence-specific oligonucleotides probes (ASO or SSO probes). Probes can 
detect single nucleotide changes which result in any of the types of 
genetic variations, so long as the exact sequence of the variable site is 
known. 
A third type of analytical method detects sequences of different lengths 
(e.g., due to an insertion/deletion of nucleotides, to nucleotide 
substitutions that change the mobility or to a change in the location of a 
restriction site) and/or different numbers of sequences (due to either 
gain or loss of restriction sites). A preferred length difference 
detection method is by gel or capillary electrophoresis. To detect changes 
in the lengths of fragments or the number of fragments due to changes in 
restriction sites, the amplified sequence must be digested with an 
appropriate restriction endonuclease prior to analysis of fragment length 
patterns. 
A fourth and most preferred type of analytical method is based on 
allele/haplotype-specific amplification to detect the presence of the 
selected allele/haplotype. In the fourth method, the locus-specific 
amplified DNA sequence is amplified with a nested primer pair specific for 
a selected allele/haplotype. Production of an amplified DNA sequence by 
the primer pair indicates the presence of the allele/haplotype. In a 
preferred embodiment of the method, each nested amplification is performed 
in a separate amplification reaction mixture so that the presence of an 
amplified DNA sequence indicates the presence of the allele/haplotype. 
Preferably, each primer pair produces an amplified DNA sequence of a 
different length and the lengths of the resultant amplified DNA sequences 
are determined to confirm the presence of the alleles/haplotypes. 
Although the analytical techniques used to recognize allele-associated 
genetic variations in the amplified DNA sequence can include use of probes 
or sequencing of the amplified DNA sequence, those methods are preferably 
limited to particular applications, such as identification of an allele 
associated with a disease. For the most part, the initial analyses are 
based on the use of amplified DNA sequence and subsequent analysis based 
on either (1) the correlation of the length of the amplified DNA sequence 
with alleles/haplotypes, (1) the production of RFLP patterns that 
correlate with alleles/haplotypes or (3) sequence-specific amplification 
where the production of an amplified DNA sequence indicated the presence 
of a selected allele/haplotype. However, sequencing or use of probes may 
be the preferred analytical method for some genetic regions. 
Primers 
Selection of primer sites 
The method of this invention is based on amplification of selected intron 
regions of genomic DNA. The methodology is facilitated by the use of 
primers that selectively hybridize to unique conserved regions of genomic 
DNA associated with a plurality of alleles of a genetic locus of interest 
and not other genetic loci. 
Thus, the sites to which primers hybridize are selected in conserved 
regions in the area to be mapped. Conserved regions are determined on the 
basis of sequences from at least two individuals. If no further sequence 
information is available, conserved regions forming the restriction site, 
clone sequence STS site or any other marker used to delineate the region 
can be used. 
When genomic DNA sequences are available, the primers are preferably 
located in conserved regions in the introns. When the only sequences 
available are cDNA sequences, the primers are located in conserved regions 
in the exons. If junctions of intron and exon sequences in the cDNA 
sequences are known, then the primer sites are preferably located near 
those junctions. 
A locus-specific primer pair contains a 5' upstream primer that defines the 
5' end of the amplified DNA sequence by hybridizing with the 5' end of the 
target sequence to be amplified and a 3' downstream primer that defines 
the 3' end of the amplified DNA sequence by hybridizing with the 
complement of the 3' end of the DNA sequence to be amplified. The primers 
in the primer pair do not hybridize with DNA of other genetic loci under 
the conditions used in the present invention. 
For each primer of the locus-specific primer pair, the primer hybridizes to 
a plurality of alleles of the DNA locus to be amplified or to its 
complement. Preferably, the primer pair amplifies all alleles of the locus 
regardless of the associated haplotypes. However, primer pairs or 
combinations thereof that specifically bind with the most common alleles 
present in a particular population group or with groups of alleles that 
share a common sequence are also contemplated. 
The amplified DNA sequence that is defined by the primers contains a 
sufficient number of non-coding region sequence nucleotides, preferably 
intron sequence nucleotides, to distinguish between alleles of an adjacent 
locus, and preferably, to identify the alleles of the locus which are 
present in the sample for all alleles of the locus, or all alleles of the 
group of alleles containing the selected sequences. In a most preferred 
embodiment, the primer-defined amplified DNA sequence contains a 
sufficient number of intron sequence nucleotides to distinguish between 
the haplotypes associated with the adjacent locus and one or more remote 
loci. 
Length of sequence 
The length of the amplified sequence which is required to include 
sufficient genetic variability to enable discrimination between all 
alleles/haplotypes of a locus bears a direct relation to the extent of the 
polymorphism of the locus (the number of alleles). That is, as the number 
of alleles and haplotypes associated with the tested locus increases, the 
size of an amplified sequence which contains sufficient genetic variations 
to distinguish each allele/haplotype increases. However, even for the HLA 
loci with numerous alleles and haplotypes, amplified DNA sequences of 
2,000 nt are sufficient. Generally, amplified DNA sequences corresponding 
to 400 to 500 nt of intron sequence nucleotides from the intron adjacent 
to the variable exon are sufficient to distinguish all the haplotypes 
associated with the loci. 
The ends of the amplified DNA sequence are defined by the primer pair used 
in the amplification. Conveniently, the primer pairs will hybridize with 
the DNA sequence of all alleles/haplotypes of the locus. Therefore, each 
primer sequence must correspond to a conserved region of the genomic DNA 
sequence. Thus, the location of the amplified sequence will, to some 
extent, be dictated by the need to locate the primers in conserved 
regions. When sufficient intron sequence information to determine 
conserved intron regions is not available, the primers can be located in 
conserved portions of the exons and used to amplify intron sequences 
between those exons. 
When appropriately-located, conserved sequences are not unique to the 
genetic locus, a second primer pair located within the amplified sequence 
produced by the first primer pair can be used to provide an amplified DNA 
sequence specific for the genetic locus. At least one of the primers of 
the second primer pair is located in a conserved region of the amplified 
DNA sequence defined by the first primer pair. The second primer pair is 
used following amplification with the first primer pair to amplify a 
portion of the amplified DNA sequence produced by the first primer pair to 
produce a locus-specific amplified DNA sequence. 
Considerations related to the genetic variation 
The type of genetic variation to be detected in the amplified DNA sequence 
also influences the location and size of the sequence. As stated 
previously, the analyses are preferably based on allele/haplotype-specific 
amplification or on the presence of genetic variations that result in a 
change in the length of the amplified DNA sequence or a change in the 
presence or location of at least one restriction site. 
For allele/haplotype-specific amplification, there are two considerations. 
The first is that the primer site for at least one of the nested primers 
is characteristic of an allele/haplotype. Those considerations are 
described in the discussion of nested primer specificity below. The second 
consideration is that preferably the amplified DNA sequence for each of 
the alleles/haplotypes differs in length. Consideration for selection of 
length differences are discussed below. 
Genetic variations that result in a difference in the length of the 
primer-defined amplified DNA sequence, referred to herein as a 
primer-defined length polymorphism (PDLP), can be used to distinguish 
between alleles/sub-allelic groups of the genetic locus. The PDLPs result 
from insertions or deletions of relatively large stretches (in comparison 
to the total length of the amplified DNA sequence) of DNA in the portion 
of the intron sequence defined by the primer pair. To detect PDLPs, the 
amplified DNA sequence is located in a region containing insertions or 
deletions of a size that is detectable by the chosen method. 
Alternatively, the length variation can be a perceived length variation 
which is due to a substitution of one or more nucleotides in the amplified 
DNA sequence that results in a change in electrophoretic mobility. This 
apparent length variation is referred to a primer-defined mobility 
variation (PDMP) and will be referred to herein as a type of PDLP. Such 
mobility differences are attributable to kinking or folding of the 
amplified DNA sequence due to particular combinations of nucleotides 
present in the sequence. Such combinations of nucleotides and the 
resultant mobility differences are well known. For example, regions rich 
in AT sequences tend to kink. 
The amplified DNA sequence should have a length which provides optimal 
resolution of length differences. For electrophoresis, DNA sequences of 
about 300 to 500 bases in length provide optimal resolution of length 
differences. However, sequences as long as 800 to 1,000 nt are also 
readily distinguishable. Under appropriate conditions, either gel 
electrophoresis or capillary electrophoresis can detect as few as three nt 
differences in sequence lengths. Preferably the length differences will be 
at least 10, more preferably 20, most preferably 50 or more, nt between 
the alleles. Therefore, preferably, the amplified DNA sequence is between 
300 to 1,000 nt and encompasses length differences of at least 3, 
preferably 10, most preferably 50 or more nt. 
PDLPs can be produced in two general ways. In the first, the primers sites 
are located in a fixed position in the sample DNA sequence and the 
sequence between the primer sites varies depending on the alleles or 
haplotypes of the locus. In another embodiment, the primer sites are 
selected at varied positions to produce an amplified DNA sequence having a 
different length for each allele/haplotype of the locus, as described 
above for allele/haplotype-specific amplification. 
When the variation to be detected is a change in a restriction site, the 
amplified DNA sequence necessarily contains at least one restriction site 
which (1) is present in one allele and not in another, (2) is apparently 
located in a different position in the sequence of at least two alleles, 
or (3) combinations thereof. The amplified sequence will preferably be 
located such that restriction endonuclease cleavage produces fragments of 
detectably different lengths, rather than two or more fragments of 
approximately the same length. 
For the method described herein, it is contemplated that use of more than 
one amplified DNA sequence and/or use of more than one analytical method 
per amplified DNA sequence may be required for highly polymorphic loci, 
loci where alleles differ by single nucleotide substitutions that are not 
unique to the allele, or when information regarding remote locus alleles 
(haplotypes) is desired. More particularly, it may be necessary to combine 
a PDLP analysis with an RFLP analysis, to use two or more amplified DNA 
sequences located in different positions, to perform multiple nested 
amplifications on the amplified DNA sequence produced by a prior nested 
amplification, or to digest one amplified DNA sequence with a plurality of 
endonucleases to provide distinctive allelic and sub-allelic patterns for 
a locus. These combinations are intended to be included within the scope 
of this invention. 
Length and sequence homology of primers 
Each locus-specific primer includes a number of nucleotides which, under 
the conditions used in the hybridization, are sufficient to hybridize with 
alleles of the locus to be amplified and to be free from hybridization 
with alleles of other loci. The specificity of the primer increases with 
the number of nucleotides in its sequence under conditions that provide 
the same stringency. Therefore, longer primers are desirable. Sequences 
with fewer than 15 nucleotides are less certain to be specific for a 
particular locus. That is, sequences with fewer than 15 nucleotides are 
more likely to be present in a portion of the DNA associated with other 
genetic loci, particularly loci of other common origin or evolutionarily 
closely related origin, in inverse proportion to the length of the 
nucleotide sequence. 
Each primer preferably includes at least about 15 nucleotides, more 
preferably at least about 20 nucleotides. The primer preferably does not 
exceed about 30 nucleotides, more preferably about 25 nucleotides. Most 
preferably, the primers have between about 20 and about 25 nucleotides. 
When two sets of primer pairs are used sequentially, with the second primer 
pair amplifying the product of the first primer pair, the primers can be 
the same size as those used for the first amplification. However, smaller 
primers can be used in the second amplification and provide the requisite 
specificity. The primers of the second primer pair can have 15 or fewer 
nucleotides. When two sets of primer pairs are used to produce two 
amplified sequences, the second amplified DNA sequence is used in the 
subsequent analysis of genetic variation and must meet the requirements 
discussed previously for the amplified DNA sequence. 
The primers preferably have a nucleotide sequence that is identical to a 
portion of the DNA sequence to be amplified or its complement. However, a 
primer having two of the first five nucleotides of the 3' end of the 
primer that differ from the target DNA sequence or its complement also can 
be used. Any nucleotides that are not identical to the sequence or its 
complement are not the 3' nucleotide of the primer. The 3' end of the 
primer preferably has at least two, preferably three or more, nucleotides 
that are complementary to the sequence to which the primer binds. Any 
nucleotides at the 3' end that are not identical to the sequence to be 
amplified or its complement will preferably not be adjacent in the primer 
sequence. More preferably, noncomplementary nucleotides in the primer 
sequence will be separated by at least two, more preferably at least 
three, nucleotides. The primers should have a melting temperature 
(T.sub.m) from about 55.degree. to 75.degree. C. Preferably the T.sub.m is 
from about 60.degree. C. to about 65.degree. C. to facilitate stringent 
amplification conditions. The degree of homology, length, T.sub.m and 
other considerations for primer selection to ensure specific hybridization 
are well known and do not constitute part of the invention. 
The primers can be prepared using a number of methods, such as, for 
example, the phosphotriester and phosphodiester methods or automated 
embodiments thereof. The phosphodiester and phosphotriester methods are 
described in Cruthers, Science 230:281-285 (1985); Brown et al, Meth. 
Enzymol., 68:109 (1979); and Nrang et al, Meth. Enzymol., 68:90 (1979). In 
one automated method, diethylphosphoramidites which can be synthesized as 
described by Beaucage et al, Tetrahedron letters, 22:1859-1962 (1981) are 
used as starting materials. A method for synthesizing primer 
oligonucleotide sequences on a modified solid support is described in U.S. 
Pat. No. 4,458,066. Each of the above references is incorporated herein by 
reference in its entirety. 
Amplification 
Once a primer pair is selected, genomic DNA is amplified to produce an 
amplified DNA sequence. The conditions and reagents for DNA amplification 
are well known. A preferred amplification method is the polymerase chain 
reaction (PCR). PCR amplification methods are described in U.S. Pat. No. 
4,683,195 (to Mullis et al, issued Jul. 28, 1987); U.S. Pat. No. 4,683,194 
(to Saiki et al, issued Jul. 28, 1987); Saiki et al, Science, 
230:1350-1354 (1985); Scharf et al, Science, 324:163-166 (1986); Kogan et 
al, New Engl. J. Med, 317:985-990 (1987) and Saiki, Gyllensten and Erlich, 
The Polymerase Chain Reaction in Genome Analysis: A Practical Approach, 
ed. Davies pp. 141-152, (1988) I.R.L. Press, Oxford. Each of the above 
references is incorporated herein by reference in its entirety. Although 
the remaining description is based on use of PCR amplification methods, 
other DNA amplification methods such as the NASBA method (Compton Nature 
350:91 1991!) can also be used. Adaptation of another DNA amplification 
method to this analysis method is within the level of skill in the art. 
Prior to amplification, a sample of genomic DNA is obtained. All nucleated 
cells contain genomic DNA and, therefore, are potential sources of the 
required DNA. For higher animals, peripheral blood cells are typically 
used rather than tissue samples. As little as 0.01 to 0.05 cc of 
peripheral blood provides sufficient DNA for amplification. Hair, semen 
and tissue can also be used as samples. Genomic DNA libraries are 
available and are readily constructed by well known methods. 
DNA isolation from nucleated cells is described by Kan et al, N. Engl. J. 
Med. 297:1080-1084 (1977); Kan et al, Nature 251:392-392 (1974); and Kan 
et al, PNAS 75:5631-5635 (1978). Each of the above references is 
incorporated herein by reference in its entirety. Extraction procedures 
for samples such as blood, semen, hair follicles, semen, mucous membrane 
epithelium and other sources of genomic DNA are well known. For plant 
cells, digestion of the cells with cellulase releases DNA. Thereafter, the 
DNA is purified as described above. 
The extracted DNA can be purified by dialysis, chromatography, or other 
known methods for purifying polynucleotides prior to amplification. 
Typically, the DNA is not purified prior to amplification. 
The amplified DNA sequence is produced by using the portion of the DNA and 
its complement bounded by the primer pair as a template. As a first step 
in the method, the DNA strands are separated into single stranded DNA. 
This strand separation can be accomplished by a number of methods 
including physical or chemical means. A preferred method is the physical 
method of separating the strands by heating the DNA until it is 
substantially (approximately 93%) denatured. Heat denaturation involves 
temperatures ranging from about 80.degree. to 105.degree. C. for times 
ranging from about 15 to 30 seconds. Typically, heating the DNA to a 
temperature of from 90.degree. to 93.degree. C for about 30 seconds to 1 
minute is sufficient. 
The primer extension product(s) produced are complementary to the 
primer-defined region of the DNA and hybridize therewith to form a duplex 
of equal length strands. The duplexes of the extension products and their 
templates are then separated into single-stranded DNA. When the 
complementary strands of the duplexes are separated, the strands are ready 
to be used as a template for the next cycle of synthesis of additional DNA 
strands. 
Each of the synthesis steps can be performed using conditions suitable for 
DNA amplification. Generally, the amplification step is performed in a 
buffered aqueous solution, preferably at a pH of about 7 to about 9, more 
preferably about pH 8. A suitable amplification buffer contains Tris-HCl 
as a buffering agent in the range of about 10 to 100 mM. The buffer also 
includes a monovalent salt, preferably at a concentration of at least 
about 10 mM and not greater than about 60 mM. Preferred monovalent salts 
are KCl, NaCl and (NH.sub.4).sub.2 SO.sub.4. The buffer also contains 
MgCl.sub.2 at about 5 to 50 mM. Other buffering systems such as hepes or 
glycine-NaOH and potassium phosphate buffers can be used. Typically, the 
total volume of the amplification reaction mixture is about 50 to 100 
.mu.l. 
Preferably, for genomic DNA, a molar excess of about 10.sup.6 :1 
primer:template of the primer pair is added to the buffer containing the 
separated DNA template strands. A large molar excess of the primers 
improves the efficiency of the amplification process. In general, about 
100 to 150 ng of each primer is added. 
The deoxyribonucleotide triphosphates dATP, dCTP, dGTP and dTTP are also 
added to the amplification mixture in amounts sufficient to produce the 
amplified DNA sequences. Preferably, the dNTPs are present at a 
concentration of about 0.75 to about 4.0 mM, more preferably about 2.0 mM. 
The resulting solution is heated to about 90.degree. to 93.degree. C for 
from about 30 seconds to 1 minute to separate the strands of the DNA. 
After this heating period the solution is cooled to the amplification 
temperature. 
Following separation of the DNA strands, the primers are allowed to anneal 
to the strands. The annealing temperature varies with the length and GC 
content of the primers. Those variables are reflected in the T.sub.m of 
each primer. The extension reaction step is performed following annealing 
of the primers to the genomic DNA. 
An appropriate agent for inducing or catalyzing the primer extension 
reaction is added to the amplification mixture either before or after the 
strand separation (denaturation) step, depending on the stability of the 
agent under the denaturation conditions. The DNA synthesis reaction is 
allowed to occur under conditions which are well known in the art. This 
synthesis reaction (primer extension) can occur at from room temperature 
up to a temperature above which the polymerase no longer functions 
efficiently. Elevating the amplification temperature enhances the 
stringency of the reaction. As stated previously, stringent conditions are 
necessary to ensure that the amplified sequence and the DNA template 
sequence contain the same nucleotide sequence, since substitution of 
nucleotides can alter the restriction sites or probe binding sites in the 
amplified sequence. 
The inducing agent may be any compound or system which facilitates 
synthesis of primer extension products, preferably enzymes. Suitable 
enzymes for this purpose include DNA polymerases (such as, for example, E. 
coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA 
polymerase), reverse transcriptase, and other enzymes (including 
heat-stable polymerases), which facilitate combination of the nucleotides 
in the proper manner to form the primer extension products. Most preferred 
is Taq polymerase or other heat-stable polymerases which facilitate DNA 
synthesis at elevated temperatures (about 60.degree. to 90.degree. C.). 
Taq polymerase is described, e.g., by Chien et al, J. Bacteriol., 
127:1550-1557 (1976). That article is incorporated herein by reference in 
its entirety. When the extension step is performed at about 72.degree. C, 
about 1 minute is required for every 1,000 bases of target DNA to be 
amplified. 
The synthesis of the amplified sequence is initiated at the 3' end of each 
primer and proceeds toward the 5' end of the template along the template 
DNA strand, until synthesis terminates, producing DNA sequences of 
different lengths. The newly synthesized strand and its complementary 
strand form a double-stranded molecule which is used in the succeeding 
steps of the process. In the next step, the strands of the double-stranded 
molecule are separated (denatured) as described above to provide 
single-stranded molecules. 
New DNA is synthesized on the single-stranded template molecules. 
Additional polymerase, nucleotides and primers can be added if necessary 
for the reaction to proceed under the conditions described above. After 
this step, half of the extension product consists of the amplified 
sequence bounded by the two primers. The steps of strand separation and 
extension product synthesis can be repeated as many times as needed to 
produce the desired quantity of the amplified DNA sequence. The amount of 
the amplified sequence produced accumulates exponentially. Typically, 
about 25 to 30 cycles are sufficient to produce a suitable amount of the 
amplified DNA sequence for analysis. 
The amplification method can be performed in a step-wise fashion where 
after each step new reagents are added, or simultaneously, where all 
reagents are added at the initial step, or partially step-wise and 
partially simultaneously, where fresh reagent is added after a given 
number of steps. The amplification reaction mixture can contain, in 
addition to the sample genomic DNA, the four nucleotides, the primer pair 
in molar excess, and the inducing agent, e.g., Taq polymerase. 
Each step of the process occurs sequentially notwithstanding the initial 
presence of all the reagents. Additional materials may be added as 
necessary. Typically, the polymerase is not replenished when using a 
heat-stable polymerase. After the appropriate number of cycles to produce 
the desired amount of the amplified sequence, the reaction may be halted 
by inactivating the enzymes or separating the components of the reaction 
or stopping thermal cycling. 
In a preferred embodiment of the method, the amplification includes the use 
of a second primer pair to perform a second amplification following the 
first amplification. The second primer pair defines a DNA sequence which 
is a portion of the first amplified sequence. That is, at least one of the 
primers of the second primer pair defines one end of the second amplified 
sequence which is within the ends of the first amplified sequence. In this 
way, the use of the second primer pair helps to ensure that any amplified 
sequence produced in the second amplification reaction is specific for the 
tested locus. That is, non-target sequences which may be copied by a 
locus-specific pair are unlikely to contain sequences that hybridize with 
a second locus-specific primer pair located within the first amplified 
sequence. 
Analysis of the Amplified DNA Sequence 
As discussed previously, the method used to analyze the amplified DNA 
sequence to characterize the allele(s) present in the sample DNA depends 
on the genetic variation in the sequence. When distinctions between 
alleles include primer-defined length polymorphisms, the amplified 
sequences are separated based on length, preferably using gel or capillary 
electrophoresis. When the analysis is based on RFLP fragment patterns, the 
amplified sequences are digested with one or more restriction 
endonucleases to produce a digest and the resultant fragments are 
separated based on length, preferably using gel or capillary 
electrophoresis. A most preferred method is an amplification-specific 
method in which the presence of a nested amplified DNA sequence indicates 
the presence of a selected an allele or haplotype. 
Each step of the various analytical methods uses procedures such as DNA 
amplification, endonuclease digestion and gel electrophoresis that are 
well known and are described below. 
Allele-or Haplotype-Specific Amplification Analysis 
Allele- or haplotype-specific amplification is a preferred analysis method 
which can be performed once 400 to 500 bp of sequence information for the 
region for about 15 to 20 individuals of the most diverse ethnic groups 
possible region is available. 
As a first step in the analysis method, a locus-specific amplified DNA 
sequence is prepared for use as a target DNA sequence for amplification by 
a nested sequence-specific primer pair, allele-specific primer pair or 
haplotype-specific primer pair. The target DNA sequence preferably 
corresponds to a portion of the genetic locus including a variable exon or 
exons just downstream from the variable exon and adjacent intron sequence 
nucleotides. The method is based on amplification of the target DNA 
sequence using a primer pair wherein at least one of the primers of the 
pair hybridizes to the target DNA sequence only when a selected sequence 
is present in the target sequence. As stated previously, the sequence 
polymorphism can be characteristic of one allele or a group of alleles of 
the genetic locus. Alternatively, the sequence polymorphism can be a 
sub-allelic variation characteristic of a haplotype associated with the 
genetic locus and one or more adjacent loci. In this way, production of an 
amplified DNA sequence indicates that the selected sequence polymorphism, 
and thus the selected sub-allelic variation, allele or group of alleles, 
is present in the target DNA. 
In one embodiment, the first amplification uses a locus-specific primer 
pair which produces a target DNA sequence irrespective of the alleles or 
haplotypes present in the sample. In another embodiment, the 
locus-specific primer pair produces a target DNA sequence only when a 
selected group of alleles having a common sequence polymorphism is present 
in the sample. The subsequent amplification of the target sequence is 
performed using primers for alleles in the group. 
The second amplification preferably contains primer pairs for sufficient 
sequence polymorphisms to determine the alleles/haplotypes in the sample. 
In one embodiment, a plurality of amplifications are performed wherein 
each amplification reaction mixture contains a single primer pair. 
Detection of the presence of amplification in a reaction mixture 
determines that the allele for which the primer pair is specific is 
present in the sample. In another embodiment, multiple DNA amplifications 
are performed in a single reaction mixture using pairs of primers wherein 
each primer pair in the reaction mixture produces an amplified DNA 
sequence having a distinguishable length from the sequence produced by 
every other primer pair in the reaction mixture. Determination of the 
length of the resultant amplified DNA sequence(s) identifies the sequence 
polymorphism(s) present in the target DNA. In a preferred embodiment, 
sufficient combinations of primer pairs are used so that the resultant 
amplified DNA sequences determine both alleles of the genetic locus 
present in the sample. 
Nested Primer Pairs 
This method is based on amplification of a target DNA sequence using a 
nested primer pair wherein the presence of amplification by the nested 
primer pair indicates that a particular sequence polymorphism is present 
in the sample. The sequence polymorphism can be characteristic of one 
allele, a group of alleles of the genetic locus or a sub-allelic variation 
characteristic of one or more individuals or characteristic of a haplotype 
associated with the genetic locus and one or more adjacent loci. 
In one preferred embodiment, the target DNA sequence is an amplified DNA 
sequence produced by a locus-specific primer pair that amplifies sample 
genomic DNA irrespective of the alleles or haplotypes present in the 
sample. In another preferred embodiment, the target DNA sequence 
encompasses groups of alleles of the locus, but not all of the alleles. 
To effect allele-specific or haplotype-specific amplification, at least one 
primer in each nested primer pair is selected so that the primer 
hybridizes to the DNA sequence only when the selected sequence 
polymorphism is present. In this way, the presence of an amplified DNA 
sequence indicates that the sequence polymorphism is present in the 
sample. This is in contrast to most prior art DNA amplification methods 
wherein primers bind to a conserved region and the resultant amplified DNA 
sequence is analyzed, usually by use of oligoprobes, for the presence of 
the polymorphism in the amplified sequence. 
At least one primer of each primer pair selectively hybridizes with a 
sequence polymorphism that is characteristic of (1) a multiplicity of 
alleles of a genetic locus but is not present in all of the alleles of the 
locus (a sequence-specific primer or SSP), (2) one allele of a genetic 
locus (an allele-specific primer or ASP), (3) one haplotype associated 
with a genetic locus and one or more adjacent loci (a haplotype-specific 
primer or HSP) or (4) a particular individual or group of individuals (an 
individual-specific primer or ISP). The other primer of the primer pair 
can be less specific, as discussed above. 
Each of the nested amplified DNA sequences are located with the target DNA 
sequence. The location of each of the nested amplified DNA sequence within 
the target DNA is selected so that one or both of the ends of the 
amplified DNA sequence include a sequence polymorphism of the desired 
specificity. That is, either the 3' end or the 5' end, or both ends, of 
the amplified DNA sequence contain a sequence polymorphism to which either 
a sequence-specific, allele-specific, haplotype-specific or 
individual-specific primer hybridizes. 
Preferably, the primers are also located such that each primer pair of in a 
reaction mixture defines an amplified DNA sequence of a different length. 
More preferably, the primers are located such that each nested primer pair 
associated with the genetic locus defines an amplified DNA sequence of a 
different length. 
The amplified DNA sequence produced by the nested primers can vary in 
length from about 50 to 700 bp, preferably from 50 to 300 bp, more 
preferably from 50 to 100 bp. Each of the nested amplified DNA sequences 
preferably also differs from each other nested amplified DNA sequence in 
the reaction mixture, preferably each nested amplified DNA sequence for 
the locus, by at least a sufficient number of nucleotides so that the 
amplified DNA sequences can be readily distinguished by gel 
electrophoresis. 
The length differences can be due to a difference in the number of 
nucleotides in the sequence (a primer-defined length polymorphism or PDLP) 
or can be an apparent length difference due to differences in mobility of 
the sequence on a gel (a primer-defined mobility polymorphism or PDMP). A 
description of gel electrophoretic analysis of PDLPs or PDMPs is described 
in detail hereinafter. 
The considerations for specific hybridization of nested primers for the 
second amplification differ somewhat from the conditions required for 
locus-specific amplification since the primers need only be sufficiently 
specific for purposes of amplifying a selected DNA sequence, the target 
sequence produced in the first amplification. The differences in the 
consideration for nested primers are well known. For the nested primers, 
each primer can vary in length from about 10 to about 30, preferably from 
15 to about 20 nt in length, most preferably about 18 nt in length. For 
each specific primer of the primer pair (an SSP, ASP, HSP, or ISP), the 3' 
end of the primer is selected to hybridize to a unique region of the 
target sequence which is characteristic of the sequence, allele haplotype 
or individual variation to be detected. 
One unique nucleotide at the 3' end is sufficient to ensure specificity 
under conditions that provide an appropriate degree of stringency for the 
amplification reaction. When possible, a location having two or three 
unique nucleotides at the 3' end of the primer site can be used. The 
primer and the primer site must be complementary for at least the 3' 
nucleotide of the primer. In addition to the 3' nucleotide, preferably at 
least two of the adjacent four nucleotides, more preferably five 
nucleotides at the 3' end of the primer are also complementary to the 
primer site sequence. Preferably, at least three nucleotides, more 
preferably five nucleotides, at the 5' end of the primer, are also 
complementary to the primer site sequence. A non-complementary region near 
the center of the primer, preferably where any non-complementary 
nucleotides are not adjacent, provides sufficient homology for specific 
amplification. 
The Analysis Method 
As described hereinbefore, the analysis method involves a first 
amplification with locus-specific primers to produce a target DNA 
sequence. The target DNA sequence is amplified with nested primer pairs 
specific for portions of the target sequence characteristic of the 
sequence, allele, haplotype or individual variation to be detected. The 
method can be performed in a number of different ways and is characterized 
by the presence of an amplified DNA sequence produced by a nested primer 
pair indicating that a sequence for which the pair is specific is present 
in the sample. 
The reagents and conditions used for DNA amplification do not differ from 
those of the locus-specific amplification. The following description of 
the method is written in terms of detecting alleles of the locus for 
purposes of clarity. The same considerations are involved in detecting 
sub-allelic variations. 
First amplification 
The first step of the analysis method is amplification of genomic DNA with 
locus-specific primers to produce a locus-specific amplified DNA sequence. 
As stated previously, the locus-specific primers produce an amplified DNA 
sequence for a group of alleles of the - locus having a common sequence 
polymorphism. The first amplification can be performed in one of two ways. 
In one embodiment, the locus-specific primer pair produces an amplified DNA 
sequence irrespective of the alleles present in the sample genomic DNA. 
The resultant locus-specific amplified DNA sequence is used as the target 
DNA for the subsequent method steps. 
In another preferred embodiment, a locus specific primer pair amplifies a 
plurality of alleles of the locus, but not all alleles of the locus. Use 
of this embodiment means that a plurality of locus-specific primer pairs 
are required to amplify all the alleles of the locus. However, the second 
amplification need only be by primers specific for the group of alleles 
having the sequence polymorphism of the locus-specific primer pair that 
produces the target DNA sequence. This method can result in a smaller 
number of total DNA amplifications being required to analyze a particular 
sample. 
Each of the locus-specific primer pairs can be present in a separate 
amplification reaction mixture. In that case, the presence of an amplified 
DNA sequence indicates the groups of the alleles in the sample. 
Alternatively, two or more locus-specific primer pairs can be present in a 
single amplification reaction mixture. In that case, each of the resultant 
amplified DNA sequences is of a distinguishable length and is 
electrophoresed to determine the group of alleles present in the sample. 
The technique of combining multiple primer pairs in a single reaction 
mixture is referred to as multiplexing. Considerations involved in 
multiplexing are described in detail below in the discussion of the second 
amplification. 
Second amplification 
The second amplification is performed using primer pairs that amplify a DNA 
sequence within the target DNA. In the second amplification, the primers 
produce amplified DNA sequences indicative of the primer pair used 
responsible for the amplification. That is, at least one primer of the 
primer pair hybridizes to a DNA sequence characteristic of an allele or 
group of alleles of the locus. Production of an amplified DNA sequence by 
a particular primer pair or group of primer pairs indicates that the 
allele is present in the sample. 
When the target sequence encompasses all alleles of the locus, an 
amplification for each allele is performed. In one embodiment, a separate 
reaction mixture is prepared for a primer pair characteristic of each 
allele of the locus. Determining the one or two reaction mixtures that 
produced an amplified DNA sequence identifies the allele(s) present in the 
sample. In a preferred embodiment, each amplified DNA sequence differs in 
length and the length of the resultant amplified DNA sequences are 
determined to confirm the alleles present in the sample. 
Alternatively, at least one of the second reaction mixtures can contain two 
or more primer pairs. By selecting combinations of primer pairs for a 
reaction mixture so that each primer pair produces an amplified DNA 
sequence having a distinguishable length from the amplified DNA sequences 
produced by every other primer pair in the reaction mixture, the primer 
pair responsible for production of the amplified DNA sequence can be 
readily identified by determining the length of the amplified DNA 
sequence. Thus the sequence polymorphism present in the target DNA can be 
readily identified by the determining the length of the amplified DNA 
sequence. 
When a plurality of primer pairs are to be used in a single reaction 
mixture, the primers of each pair are selected so that the length of the 
amplified DNA sequence is distinguishable from the lengths of all other 
amplified DNA sequences produced by other primer pairs present in the 
reaction mixture. Each primer of the pair can be specific for the selected 
allele. Alternatively, two or more primer pairs in the reaction mixture 
can share a common primer. When one primer pair in a reaction mixture 
utilizes a common primer, conveniently, all the primer pairs in the 
reaction mixture utilize the common primer. In that case, the 
allele-specific primer for each of the primer pairs will be selected at 
locations that are sufficiently far from the other allele-specific primers 
to be distinguishable by gel electrophoresis. 
In a preferred embodiment, only one amplified DNA sequence is produced for 
each allele. That is, preferably, there is one allele-specific primer pair 
for each allele of the locus. For example, when several allele-specific 
primer pairs are used, the primers for each pair are selected so that only 
one amplified DNA sequence is produced when the allele is present in the 
target DNA. However, patterns of sequence-specific amplified DNA sequences 
which are characteristic of an allele when occurring together are also 
contemplated. 
In another embodiment, one or more of the nested primer pairs in the second 
reaction mixture are sequence-specific primers and amplify a group of 
alleles of the locus. An additional amplification using nested primers to 
amplify the amplified DNA sequence produced by second amplification is 
used to determine the alleles within the second amplified DNA sequence. 
The analysis method described above for alleles of the locus can be readily 
applied by one of ordinary skill in the art to analysis of suballelic 
polymorphisms, particularly haplotypic polymorphisms, or to use of groups 
of sequence-specific primer that produce patterns of amplified DNA 
sequences that characterize the alleles/haplotypes or that are amplified 
with nested primers to determine the alleles/haplotypes. 
Production of RFLP Fragment Patterns 
Restriction endonucleases 
A restriction endonuclease is an enzyme that cleaves or cuts DNA 
hydrolytically at a specific nucleotide sequence called a restriction 
site. Endonucleases that produce blunt end DNA fragments (hydrolysis of 
the phosphodiester bonds on both DNA strands occur at the same site) as 
well as endonucleases that produce sticky ended fragments (the hydrolysis 
sites on the strands are separated by a few nucleotides from each other) 
can be used. 
Restriction enzymes are available commercially from a number of sources 
including Sigma Pharmaceuticals, Bethesda Research Labs, 
Boehringer-Manheim and Pharmacia. As stated previously, a restriction 
endonuclease used in the present invention cleaves an amplified DNA 
sequence of this invention to produce a digest comprising a set of 
fragments having distinctive fragment lengths. In particular, the 
fragments for one allele/haplotype of a locus differ in size from the 
fragments for other alleles/haplotypes of the locus. The patterns produced 
by separation and visualization of the fragments of a plurality of digests 
are sufficient to distinguish allelic and sub-allelic patterns for the 
locus. More particularly, the endonucleases are chosen so that by using 
one or more digests of the amplified sequence, the alleles of a locus can 
be distinguished. 
In selecting an endonuclease, the important consideration is the number of 
fragments produced for amplified sequences of the various alleles of a 
locus. More particularly, a sufficient number of fragments must be 
produced to distinguish between the alleles haplotypes. However, the 
number of fragments must not be so large or so similar in size that a 
pattern that is not distinguishable from those of other haplotypes by the 
particular detection method is produced. This selection is preferably 
performed by analyzing representative sequences and determining useful 
restriction endonucleases for the sequence rather than by empirically 
combining different endonucleases with the amplified DNA sequences and 
evaluating the sufficiency of the resultant patterns. 
One of ordinary skill can readily determine whether an endonuclease 
produces RFLP fragments having distinctive fragment lengths. The 
determination can be made experimentally by cleaving an amplified sequence 
for each allele with the designated endonuclease in the invention method. 
The fragment patterns can then be analyzed. Preferably, the sequences are 
analyzed and an endonuclease restriction sites present in the sequences of 
the locus that produce fragments characteristic of alleles/haplotypes are 
selected. Distinguishable patterns will be readily recognized by 
determining whether comparison of two or more digest patterns is 
sufficient to detect differences between the patterns of the alleles. Such 
comparisons can be made by producing the fragments and separating the 
fragments on a gel. Alternatively, the fragments produced by an 
endonuclease can be determined by analyzing known sequences to determine 
the lengths of the fragments. 
Production of RFLP fragments 
Following amplification, the amplified DNA sequence is combined with an 
endonuclease that cleaves or cuts the amplified DNA sequence 
hydrolytically at a specific restriction site. The combination of the 
endonuclease with the amplified DNA sequence produces a digest containing 
a set of fragments having distinctive fragment lengths. U.S. Pat. No. 
4,582,788 (to Erlich, issued Apr. 15, 1986) describes an HLA typing method 
based on restriction length polymorphism (RFLP). That patent is 
incorporated herein by reference in its entirety. 
In a preferred embodiment, two or more aliquots of the amplification 
reaction mixture having approximately equal amounts of DNA per aliquot are 
prepared. Conveniently about 5 to about 10 .mu.l of a 100 .mu.l reaction 
mixture is used for each aliquot. Each aliquot is combined with a 
different endonuclease to produce a plurality of digests. In this way, by 
using a number of endonucleases for a particular amplified DNA sequence, 
locus-specific combinations of endonucleases that distinguish a plurality 
of alleles of a particular locus can be readily determined. Following 
preparation of the digests, each of the digests can be used to form RFLP 
patterns. Preferably, two or more digests can be pooled prior to pattern 
formation. 
Alternatively, two or more restriction endonucleases can be used to produce 
a single digest. The digest differs from one where each enzyme is used 
separately and the resultant fragments are pooled since fragments produced 
by one enzyme may include one or more restriction sites recognized by 
another enzyme in the digest. Patterns produced by simultaneous digestion 
by two or more enzymes will include more fragments than pooled products of 
separate digestions using those enzymes and will be more complex to 
analyze. 
The digestion of the amplified DNA sequence with the endonuclease can be 
carried out in an aqueous solution under conditions favoring endonuclease 
activity. Typically the solution is buffered to a pH of about 6.5 to 8.0. 
Mild temperatures, preferably about 20.degree. C. to about 45.degree. C., 
more preferably physiological temperatures (25.degree. to 40.degree. C.), 
are employed. Restriction endonucleases normally require magnesium ions 
and, in some instances, cofactors (ATP and S-adenosyl methionine) or other 
agents for their activity. Therefore, a source of such ions, for instance 
inorganic magnesium salts, and other agents, when required, are present in 
the digestion mixture. Suitable conditions are described by the 
manufacturer of the endonuclease and generally vary as to whether the 
endonuclease requires high, medium or low salt conditions for optimal 
activity. 
The amount of DNA in the digestion mixture is typically in the range of 1% 
to 20% by weight. In most instances 5 to 20 .mu.g of total DNA digested to 
completion provides an adequate sample for production of RFLP fragments. 
Excess endonuclease, preferably one to five units/.mu.g DNA, is used. 
The set of fragments in the digest is preferably further processed to 
produce RFLP patterns which are analyzed. If desired, the digest can be 
purified by precipitation and resuspension as described by Kan et al, PNAS 
75:5631-5635 (1978), prior to additional processing. That article is 
incorporated herein by reference in its entirety. 
Once produced, the fragments are analyzed by well known methods. 
Preferably, the fragments are analyzed using electrophoresis. Gel 
electrophoresis methods are described in detail hereinafter. Capillary 
electrophoresis methods can be automated (as by using Model 207A 
analytical capillary electrophoresis system from Applied Biosystems of 
Foster City, Calif.) and are described in Chin et al, American 
Biotechnology Laboratory News Edition, December, 1989. 
Electrophoretic Separation of DNA Fragments 
Electrophoresis is the separation of DNA sequence fragments contained in a 
supporting medium by size and charge under the influence of an applied 
electric field. Gel sheets or slabs, e.g. agarose, agarose-acrylamide or 
polyacrylamide, are typically used for analysis of nucleotide sequencing. 
The electrophoresis conditions effect the desired degree of resolution of 
the fragments. A degree of resolution that separates fragments that differ 
in size from one another by as little as 10 nucleotides is usually 
sufficient. Preferably, the gels will be capable of resolving fragments 
which differ by 3 to 5 nucleotides. However, for some purposes, 
discrimination of sequence differences of at least 100 nt may be 
sufficient for the analysis. 
Preparation and staining of analytical gels is well known. For example, a 
3% Nusieve 1% agarose gel which is stained using ethidium bromide is 
described in Boerwinkle et al, PNAS, 86:212-216 (1989). Detection of DNA 
in polyacrylamide gels using silver stain is described in Goldman et al, 
Electrophoresis, 3:24-26 (1982); Marshall, Electrophoresis, 4:269-272 
(1983); Tegelstrom, Electrophoresis, 7:226-229 (1987); and Allen et al, 
BioTechniques 7:736-744 (1989). The method described by Allen et al, using 
large-pore size ultrathin-layer, rehydratable polyacrylamide gels stained 
with silver is preferred. Each of those articles is incorporated herein by 
reference in its entirety.

This invention is further illustrated by the following specific but 
non-limiting examples. Temperatures are given in degrees Centigrade and 
concentrations as weight percentages unless otherwise specified. 
Procedures that are constructively reduced to practice are described in 
the present tense, and procedures that have been carried out in the 
laboratory are set forth in the past tense. 
EXAMPLE 1 
Analysis of the HLA DQA Locus 
The haplotypes of the HLA DQA1 locus were analyzed as described below. DNA 
from individuals of each known haplotype of the DQA1 locus was evaluated. 
Approximately 1 .mu.g of sample DNA was combined in a total volume of 100 
.mu.l with a primer pair (1 .mu.g of each primer), dNTPs (2.5 mM each) and 
2.5 units of Taq polymerase in amplification buffer (50 mM KCl; 10 mM 
Tris-HCl, pH 8.0; 2.5 mM MgCl.sub.2 ; 100 .mu.g/ml gelatin) to form 
amplification reaction mixtures. 
The sequences of the primers were: 
SGD 001--5' TTCTGAGCCAGTCCTGAGA 3'(SEQ ID NO: 1); and 
SGD 003--5' GATCTGGGGACCTCTTGG 3'(SEQ ID NO: 2). 
These primers hybridize to sequences about 500 bp upstream from the 5' end 
of the second exon and 50 bp downstream from the second exon and produce 
amplified DNA sequences in the 700 to 800 bp range. Each primer was 
synthesized using an Applied Biosystems model 308A DNA synthesizer. 
The amplification procedure used thirty cycles of 94.degree. C. for 30 
seconds, 60.degree. C. for 30 seconds, and 72.degree. C. for 60 seconds. 
Following amplification, the amplified DNA sequences were electrophoresed 
on a 4% polyacrylamide gel to determine the PDLP type. In this case, 
amplified DNA sequences for the eight alleles produced five different 
length PDLP sequences, (demonstrating the presence of at least 5 
haplotypes). Subsequent enzyme digestion used to produce RFLP patterns 
distinguished additional allelic and sub-allelic (haplotypic) patterns. 
The amplified DNA sequences were aliquoted and separately digested using 
the restriction enzymes AluI, DdeI and MboII (Bethesda Research 
Laboratories). The digestion was performed by mixing 5 units (1 .mu.l) of 
enzyme with 10 .mu.l of the amplified DNA sequence (between about 0.5 and 
1 .mu.g of DNA) in the enzyme buffer provided by the manufacturer 
according to the manufacturer's directions to form a digest. The digest 
was then incubated for 2 hours at 37.degree. C. for complete enzymatic 
digestion. 
The products of the digestion reaction were mixed with approximately 0.1 
.mu.g of "ladder" nucleotide sequences (nucleotide control sequences 
beginning at 123 bp in length and increasing in length by 123 bp to a 
final size of about 5,000 bp; available commercially from Bethesda 
Research Laboratories, Bethesda Md.) and electrophoresed using a 4% 
horizontal ultra-thin polyacrylamide gel (E-C Apparatus, Clearwater Fla.). 
The bands in the gel were visualized (stained) using silver stain 
technique Allen et al, BioTechniques 7:736-744 (1989)!. 
PDLP groups and fragment patterns for each of the DQA1 haplotypes with each 
of the three endonucleases are illustrated in Table 1. 
##STR1## 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Homo sapiens 
(D) DEVELOPMENTAL STAGE: Adult 
(viii) POSITION IN GENOME: 
(A) CHROMOSOME/SEGMENT: 6 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
TTCTGAGCCAGTCCTGAGA19 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Homo sapiens 
(viii) POSITION IN GENOME: 
(A) CHROMOSOME/SEGMENT: 6 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GATCTGGGGACCTCTTGG18 
__________________________________________________________________________