Linkage analysis of genes with diseases using difference spectrum analysis

The present invention is directed to a method for rapidly analyzing whether known genetic markers which are found in various lengths in the population, e.g., those containing (CA).sub.n repeats, are associated with a disease of interest. The method involves using polymerase chain reactions to amplify the DNA in the marker regions and comparing the amplified DNA lengths seen in the normal population with those seen in an affected population of persons. The method involves a pooling of DNA samples from normal patients to average out the normal marker genotype found in the population and also involves a pooling of DNA from affected individuals to give a summing effect to give a stronger signal. The amplified DNA fragments are labeled with two distinguishable markers such as two different colored fluorescent markers, one used to label the amplified DNA from the normal population and the other to label the amplified DNA from the affected population. The amplified products from the normals and the affecteds are pooled, run on a sequencing gel, and a difference spectrum is calculated. Markers which are not associated with the disease will result in a zero or near zero difference spectrum whereas a marker which is associated with the disease will result in a difference spectrum with marked peaks.

BACKGROUND OF THE INVENTION 
Advances in the techniques of molecular biology are leading to the 
development of massive amounts of data concerning the sequence of the 
genomes of many organisms including humans. Genes are being sequenced with 
increasing rapidity once they are located, with plans well underway to 
sequence completely the complete genomes of several organisms including 
humans. Once genes associated with a disease are located specifically on a 
chromosome and fully analyzed by DNA sequencing, it may be possible to 
correct genetic defects found in these genes by gene therapy techniques. 
Gene therapy is still in its infancy, but there have been some promising 
results produced already. A prerequisite for performing gene therapy is 
the identification and sequence analysis of a gene associated with the 
disease in question. Locating genes specifically associated with a disease 
is an extremely complex process. Much of this complexity is a result of 
the very large size of genomes. The haploid human genome consists of 
approximately 3 billion nucleotides spread across 23 different chromosomes 
(24 if the X and Y are considered separately, and 25 if one includes the 
mitochondrial chromosome). Locating a single gene associated with a 
specific disease presently can take years of research by large groups of 
scientists working in concert. 
Presently, efforts are well underway to sequence the complete human genome. 
Hopes are to finish this sequencing within another 10 years. Nevertheless, 
even if the complete sequence of the human genome is determined, these 
data alone cannot match a gene with a disease. Such a process must be done 
by chromosome mapping. Typically, one tries to find large families 
affected with the disease or affected sib pairs. By studying many markers 
spread throughout the genome and determining which markers are 
consistently seen with the disease it is possible to locate a chromosomal 
region likely to contain the gene of interest. Once the chromosomal region 
has been narrowed to a relatively small region, e.g., 1-5 million base 
pairs, sequencing studies may take over, this region of DNA being 
sequenced to determine normal sequence and comparing this normal sequence 
to the sequence determined from affected individuals. An alternative to 
sequencing genomic DNA is to identify candidate genes by strategies which 
look for transcripts, e.g., screening cDNA libraries or using hybrid 
selection. If a gene is found which consistently is mutated in affected 
individuals then it is highly likely that the gene is associated with the 
disease. Useful sequencing methods for DNA were developed in the 1970s and 
have become highly automated in the succeeding years. DNA sequencing is no 
longer the rate limiting step in finding a gene associated with a disease. 
Instead, the hard step today is to map physically a putative gene to a 
chromosomal location. 
Various techniques have been developed for genetic linkage analysis. Given 
a set of informative families, genetic markers are essential for linking a 
disease to a region of a chromosome. Such markers include restriction 
fragment length polymorphisms (RFLPs) (Botstein, D. et al., Am. J. Hum. 
Genet. 32:314-331 (1980), markers with a variable number of tandem repeats 
(VNTRs) (Jeffreys, A. et al., Nature 314:67-73 (1985); Nakamura et al., 
Science 235:1616-1622 (1987)), and an abundant class of DNA polymorphisms 
based on short tandem repeats (STRs), especially repeats of CpA (Weber and 
May, Am. J. Hum. Genet. 44:388-396 (1989); Litt et al., Am. J. Hum. Genet. 
44:397-401 (1989)). These STRs are found in microsatellite DNA. Such 
markers tend to consist of small repeats, for example (CA).sub.n where 
n=10-30. The definition of a genetic marker here is that the genetic 
marker includes the repeat DNA as well as surrounding unique regions of 
DNA. Other dinucleotide repeats, trinucleotide repeats, or even larger 
repeats are seen and can be used. These microsatellite repeat markers are 
useful because they are scattered across the genome at approximately every 
100 kilobases. A useful marker is one which is polymorphic in the total 
population, e.g., if a (CA).sub.n marker is used, it is not really useful 
if 99% of the population has the same number of repeats. A CA marker 
located near gene A may have 20 CA repeats in it. If 99% of the population 
all has 20 repeats in the marker it is useless as a marker. Contrarily, if 
instead 10% of the population has 18 repeats, 5% has 19 repeats, 15% has 
20 repeats, 30% has 21 repeats, 30% has 22 repeats, and 10% has 23 
repeats, the marker may be quite useful. The marker is heterogeneous with 
six possible repeats. This marker can be used to study gene A. If in a 
large family with many affected persons, it is seen that all of the 
affected persons are homozygous or hemizygous for 23 repeats of the CA 
whereas the unaffected members are never homozygous for the 23 repeat form 
of the CA satellite marker, this is very suggestive that gene A (or 
another gene located relatively nearby) is associated with the disease. 
SUMMARY OF THE INVENTION 
The present invention which is disclosed will prove useful in increasing 
the rate at which a specific gene can be matched up with a marker and thus 
localized to a small region of a chromosome. The technique results in an 
increase of 1 to 2 orders of magnitude for this process. It uses known 
microsatellite markers, e.g., those containing dinucleotide repeats of 
(CA).sub.n. Thousands of such markers are scattered throughout the human 
genome, each with unique sequence on both sides of the dinucleotide 
repeats. The invention utilizes a process wherein primers are made 
complementary to each side of the markers to be studied and a polymerase 
chain reaction is performed. Two sets of reactions are carried out, one in 
which the primers are labeled with a fluorescent marker, e.g., blue, and 
the other in which the primers are labeled with a different fluorescent 
marker, e.g., red. One color primer is used for PCR for persons affected 
with the disease (affected), the other marker is used for PCR for a group 
of persons not affected with the disease (normal). The PCR reactions are 
carried out on several affected and several normal persons, with the DNA 
samples being amplified separately or by the pooling of several affected 
samples and the pooling of several normal samples. Several PCRs using 
several different sets of primers may all be performed together in a 
single tube and thereby increase the throughput, but this is not 
necessary. After completing the PCRs, the amplified DNAs are 
electrophoresed on a gel to separate them by size. Many samples are run in 
a single lane at one time. It is this last aspect of the invention which 
most allows for the increased rapidity in locating genes as compared to 
earlier methods. It is most convenient to run these on an automated 
sequencing apparatus such as an ABI 373. The intensities of the blue and 
red are recorded either as the electrophoresis occurs or following 
electrophoresis. The blue and red spectra are then compared and one is 
subtracted from the other after appropriate mobility corrections and 
scaling factors are applied. If the particular marker of interest is not 
associated with the disease, then the affected and the normal PCR products 
contain the same relative amounts of each repeat for the marker. The 
difference spectrum will approximate a relatively flat line. If, however, 
the marker is associated with the disease, it is likely one multiple of 
the dinucleotide will prevail in the affecteds. This will appear as a peak 
in the difference spectrum. If several markers had been mixed together, 
when a difference spectrum peak is observed, those markers can be 
reanalyzed separately. By performing the PCR on DNA from several persons 
(or other organisms) at one time the signals are averaged and results in 
an improved signal to noise ratio because the background noise will be 
averaged out. By not only pooling DNA from many individuals to decrease 
noise, but also by possibly pooling the primers for markers into a single 
PCR reaction and especially by pooling many reaction products to be run 
together on a single lane of a gel, there results a dramatic increase in 
the rate of correlating a marker with a disease.

DESCRIPTION OF THE INVENTION 
The present invention is directed to a method to locate more rapidly the 
chromosomal location of a gene associated with a disease. In general, the 
invention combines two techniques to accomplish this increase in speed 
compared to presently used methods of gene mapping. One technique is to 
pool DNA from several individuals to average out background noise and thus 
decrease the possibility of false positive results which could lead one 
astray. The second technique is to analyze several markers together in 
single lanes of a gel rather than individually, again leading to more 
rapid analysis. 
More specifically, the method of the present invention comprises: (a) 
selecting several genetic markers; (b) preparing two sets of primer pairs 
each set complementary to each genetic marker; (c) purifying normal DNA 
from a population of normal; (d) purifying mutated DNA from a population 
of affected persons; (e) performing polymerase chain reactions on said 
normal DNA and on said mutated DNA using the two sets of primers to 
produce amplified DNA fragments; (f) pooling the amplified DNA fragments 
and electrophoresing them on a gel; (g) scanning the amplified DNA 
fragments to measure a first signal wherein the first signal indicates 
relative amounts of each size of amplified normal DNA fragment and to 
measure a second signal wherein the second signal indicates relative 
amounts of each size of amplified mutated DNA; (h) determining a 
difference spectrum between the first and second signals; and (i) 
examining the difference spectrum, wherein a difference spectrum showing a 
difference of close to zero throughout indicates no association of the 
markers with the disease and wherein a peak in the difference spectrum 
indicates a positive association of one or more markers with the disease. 
The two sets of primer pairs are prepared such that each end of a genetic 
marker is complementary to one primer of each primer pair such that each 
pair of primers will amplify a genetic marker in a polymerase chain 
reaction. At least one primer of each pair from a first set of primer 
pairs is labeled with a first marker and at least one primer of each pair 
from a second set of primer pairs is labeled with a second marker wherein 
the first marker is distinguishable from the second marker. The primers 
labeled with the first marker are used in the polymerase chain reactions 
with the normal DNA and the primers labeled with the second marker are 
used in the reactions with the mutated DNA. The difference spectrum is 
determined by normalizing the first signal with the second signal to 
produce a scaled first signal and a scaled second signal and subtracting 
the scaled first signal from the scaled second signal. The normal DNA is 
preferably pooled prior to amplification. The affected persons are 
preferably blood relatives. 
A common method of gene mapping as presently performed can be outlined as 
follows: several gene markers which are fairly heterogeneous, often due to 
the presence of dinucleotide repeats with varying numbers of the repeat, 
are analyzed in persons with and without a disease. It is most useful if 
rather large families can be found for this purpose. Studying sib pairs is 
also a useful technique but not quite so good as having large families to 
study. Commonly, the marker region of each individual family member is 
amplified by PCR and the amplified region is sequenced by DNA sequencing 
to determine the number of dinucleotide repeats in the individual. For 
example, a single heterogeneous marker may have anywhere from 18-23 
dinucleotide repeats in the general population. For this example assume 
the relative frequencies are 5, 10, 15, 30, 30, 5 and 5% respectively for 
the 18, 19, 20, 21, 22 and 23 repeats. If the normal and affected 
individuals being tested show the same relative frequency of each type of 
repeat then it is unlikely that the marker is associated with the disease. 
However, if the affected population fairly consistently shows, e.g., only 
the 23 repeat version whereas the normal members of the family show the 
other repeats as well, there is a high likelihood that the marker is 
located near the gene causing the disease. In practice it is difficult to 
determine such an association unless the marker is located fairly close to 
the gene of interest. Crossing over of chromosomes leads to much of this 
complication. It is common to analyze many, many markers before any 
promising leads are found. Analyzing every individual in a family one by 
one is very laborious. It also can lead to complications such as in the 
case where, in an affected person, crossing over has occurred between the 
gene of interest and a marker which is relatively close to a gene of 
interest. In such a case it will appear that the marker is not located 
near the gene of interest. For a recessive gene in a person in which such 
a crossing over has occurred, such a person who has become hemizygous for 
the gene will have the recessive phenotype but will be seen as having a 
marker found in normal individuals rather than the marker found in 
affected individuals. This greatly confuses the data. A method to decrease 
such false results will be very useful. 
The present invention decreases the complications introduced by crossing 
over between chromosomes. It does this by pooling the DNA of many 
individuals who are to be tested. This effectively decreases the 
background noise of the obtained data by summing the results seen for many 
affected individuals and prevents one from following up false positive 
results and therefore prevent a lot of unnecessary work going in the wrong 
direction. The present invention also uses a multiplexing or parallel 
processing technique by analyzing several markers, as many as 10-100, 
together in single lanes on a gel to speed up greatly the screening of 
markers to find those which are truly associated with the gene of 
interest. By combining these two pooling techniques and by using a method 
which does not require sequencing of individual marker regions but simply 
looks at the sizes of amplified products and the relative percentage of 
each size both in normal and in affected individuals, and then comparing 
these two sets of data via a simple difference spectrum, a very rapid 
screening of markers can be accomplished. This method results in an 
increase of 1 to 2 orders of magnitude compared to other methods which are 
presently used. 
To perform the method of the invention, DNA is obtained from both normal 
and affected persons. The procedure gives better results as the number of 
individuals from whom DNA is obtained increases. It is also best to match 
the population of normal and affected persons as nearly as possible. Using 
blood relatives is the best approach if possible. Genetic markers to be 
tested are chosen. Primer pairs complementary to the genetic markers are 
made to be used in performing polymerase chain reactions. At least one of 
each primer pair is labeled with a marker, preferably a fluorescent 
marker. Two sets of primer pairs must be prepared, one of which has a 
first label and the other which has a second label. One set will be used 
to perform PCR with normal DNA as the template and the other set will be 
used to perform PCR with the affected (mutated) DNA as the template. The 
two labels must be distinguishable. Labels such as fluorescently labeled 
nucleotides which are commonly used for automated sequencing are 
preferred. 
The DNA from the normal individuals will normally be pooled prior to 
performing PCR although the samples may be amplified individually. The DNA 
from the affected individuals is usually amplified individually rather 
than being pooled, although all of the individual affected DNA samples may 
be pooled prior to amplification. If this pooling is done prior to 
amplification some of the individual samples of DNA may be very 
underrepresented in the amplified DNA and not be seen. In performing the 
PCR on either the affected or normal DNA samples it is most preferable to 
perform individual reactions using only a single primer pair for each 
reaction and then pooling the PCR products. It is best to pool 
stoichiometric amounts of each amplified product. To reduce the number of 
reactions it is possible to pool primers in PCRs, e.g. using 3 primers 
pairs in a single reaction, but again this may result in 
underrepresentation of products from some primer pairs and data may be 
lost as a result. If this pooling of primer pairs is done, the primer 
pairs should be designed to have nearly identical T.sub.m s. 
After performing PCR, many samples are pooled and run together on a single 
lane of a sequencing gel. Ten to one hundred individual marker reactions 
may be mixed in a single lane. The amplified normal DNA and amplified 
affected DNA are mixed together to be run on the same lane. To measure the 
amplified products it is most preferable to scan the gel at the emission 
maxima of the two fluorescent labels. The intensity spectra of the two 
labels are recorded, normalized to one another and a difference spectrum 
is calculated. If none of the markers is associated with the disease of 
interest, the difference spectrum should be a nearly flat line. If one of 
the markers is associated with the disease, this marker will cause a peak 
in the difference spectrum. When a peak is observed it is necessary to 
redo the experiment using the pooled markers individually to determine 
which marker was responsible for the peak. Because it is rare to find a 
marker associated with a disease most experiments will not show a peak and 
the markers used in such experiments need not be tested individually. The 
ability to load as many as 100 markers into a single lane allows one to 
quickly screen and eliminate many markers. Further, by mixing markers in a 
single lane which give a wide range of sizes of amplified DNA, it is 
possible to narrow the choice of which markers gave a peak to only those 
markers producing amplified DNA of the size at which the peak occurred. 
The present invention is described with reference to the following 
Examples, which are offered by way illustration and are not intended to 
limit the invention in any manner. Standard techniques well known in the 
art or the techniques specifically described below are utilized. 
EXAMPLE 1 
In this example, a single microsatellite maker is analyzed. It contains the 
dinucleotide marker (CA).sub.n where n=10-15. Primers corresponding to the 
unique DNA sequences on each side of the repeat region are used in a 
polymerase chain reaction to amplify the DNA from pools of individuals. 
The primers are selected such that an amplified DNA fragment is 60 bases 
in length when n=10 and 70 bases in length when n=15. DNA is isolated from 
100 normal persons and pooled in stoichiometric amounts, and DNA is 
isolated from 10 affected blood relatives from the same family. This 
latter DNA may be pooled prior to amplification, but it is preferable to 
amplify the samples separately to assure no sample is underrepresented. If 
amplified separately, the amplified products are pooled prior to gel 
electrophoresis. The individuals chosen for the normal DNA samples are 
matched as closely as possible in genetic background to the affected 
individuals. Using members of the same family is the most preferable 
option. If this is not possible, then other factors are looked at. For 
example, if the affected population is African-American then the normal 
population should be African-American, if the affected population is 
Scandinavian then the normal population should be Scandinavian, etc. The 
two sets of DNA are each amplified by polymerase chain reactions. One 
primer used for the normal individuals is labeled with a blue fluorescent 
marker, and one primer used for the affected individuals is labeled with a 
red fluorescent marker. Following PCR, the two samples (normal and 
affected) are combined and electrophoresed in a single lane of a 
sequencing gel apparatus, for example the ABI Model 373. The amplified DNA 
sequences will vary from 60 bases in length up to 70 bases in length, with 
lengths of 60, 62, 64, 66, 68 and 70 bases being possible. As the samples 
are electrophoresed, the fluorescence emission is measured at the peak 
intensities of the two labels. The intensities are normalized to each 
other, and a difference spectrum is calculated. If the difference spectrum 
is a relatively flat line, this indicates that each of the two sets of 
amplified DNA have roughly the same percentage of each possible size of 
DNA products and no correlation between the marker and the disease is 
established. If the difference spectrum shows a marked peak in the 
difference spectrum, for example, at a size of 66 base pairs, this 
indicates that marker is closely associated with the disease under study. 
This results from the likely fact that the genetic mutation originated in 
a single ancestor who had the marker with 13 dinucleotide repeats which 
gives the 66 base amplified product. Thus all descendants who received the 
mutated gene will also receive the 66 base marker, unless of course there 
had been crossing over between the gene of interest and the marker. The 
closer the marker is to the gene of interest the cleaner will be the 
results. By pooling DNA from many individuals, such crossing over effects 
are diluted out. 
EXAMPLE II 
The first example just given was for a simple case of using only a single 
marker. The process of screening for a useful marker can be dramatically 
increased by using several markers simultaneously throughout all of the 
steps of the process. For this example, once again DNA is isolated from 
100 blood relatives and pooled and DNA is isolated from 10 affected 
individuals from the same family and pooled. Here 3 sets of primer pairs 
will be utilized for PCR. It is preferable to choose markers and primers 
which will give a reasonable separation of DNA bands with minimal overlap 
when electrophoresed. Marker 1 may be a microsatellite region with a 
(CA).sub.n repeat with n=10-13. Marker 2 may be similar with n=15-19, and 
marker 3 may have n=21-25. If each primer used is 15 nucleotides long, 
then marker 1 will give amplified DNA of sizes 50-56 base pairs, marker 2 
will result in amplified DNA of 60-68 base pairs, and marker 3 will give 
sizes of 72-80 base pairs. In this scenario there is no overlap between 
any of the resulting products and the 3 markers can easily be 
distinguished on a single lane. As in Example I, the pooled DNAs are 
amplified via PCR except here the 3 sets of primers are used and all the 
resulting products are mixed together and electrophoresed in a single lane 
of a gel, the emission spectra are analyzed, and a difference spectrum is 
calculated. Again, a flat difference spectrum is indicative of a lack of 
correlation between the marker and the disease, whereas a peak in the 
difference spectrum indicates a correlation. 
EXAMPLE III 
This example will illustrate the simultaneous analysis of several markers 
for the situation in which the lengths of the amplified DNA fragments 
resulting from different marker regions overlap in size. Assume the case 
of 10 distinct markers, named A-J, each with (CA).sub.n dinucleotide 
repeats. Further assume the case for which each marker is examined by 
performing PCR using primers which are all 15 nucleotides in length and 
immediately abut the (CA).sub.n repeat region. Note that in practice the 
primers need not immediately abut the repeat region but can be moved 
farther away purposely to adjust the lengths of the amplified DNA 
fragments if such will be useful in achieving better separation of 
different markers. On sequencing gels, bands of DNA as large as 300 
nucleotides can easily be distinguished from bands of DNA of length 301 
nucleotides. Therefore primers may be moved quite some distance away from 
the repeat region to give products as large as approximately 300 
nucleotides; there is no necessity of using only short products as in the 
50-100 nucleotide length. This allows for running several samples together 
in a single lane and yet having minimal overlap between them. Lengths can 
also be adjusted somewhat by selection of the lengths of the primers used 
for the PCR. Table I lists the repeat size ranges and also the size ranges 
of the amplified products. 
TABLE I 
______________________________________ 
Range of Lengths of 
Marker Range of Repeats 
Amplified Products 
______________________________________ 
A 10-15 50-60 
B 10-15 50-60 
C 16-21 62-72 
D 17-24 64-78 
E 19-26 68-82 
F 19-28 68-86 
G 22-26 74-82 
H 23-28 76-86 
I 23-29 76-88 
J 25-30 80-90 
______________________________________ 
As in the previous example, two sets of reactions are performed, one in 
which stoichiometric amounts of DNA from 100 normal blood relatives are 
mixed for the PCR and one in which stoichiometric amounts of DNA from 10 
affected blood relatives from the same family are used. One primer from 
each pair is labeled with either a blue fluorescent marker (for use with 
the normals) or a red fluorescent marker (for use with the affecteds). 
PCRs are performed using each set of primers in individual reactions. 
(Note: It may be possible to mix all the primers into a single PCR. If one 
desires to do this, balancing greater speed against the chance of missing 
a marker, it is best to choose primers which will all work equally well 
under a single set of PCR conditions, e.g., one will want to choose all 
sets of primers to have equal T.sub.m s. Some minor variations between the 
primer efficiency is acceptable.) After performing the polymerase chain 
reactions the two sets of amplified DNA (that from the normals and the 
affecteds) is mixed and run on a sequencing gel apparatus. The emission 
spectra are recorded during the electrophoresis. The two spectra are then 
normalized and a difference spectrum is computed. 
In this example it is seen that markers A and B will give amplified 
products which completely overlap in size range and do not overlap any of 
the other amplified products. Each marker appears in 6 different sizes in 
the normal population. Table II lists the percent of each size seen in the 
normals. 
TABLE II 
______________________________________ 
Expected % of Each Product in Normals 
Size of Amplified 
Product % of Product for A 
% of Product for B 
% A + B 
______________________________________ 
50 5 10 7.5 
52 10 10 10.0 
54 20 30 25.0 
56 50 25 37.5 
58 5 10 7.5 
60 10 15 12.5 
______________________________________ 
When the amplified products are electrophoresed, if one looks solely at the 
blue spectrum produced from the normals in the range of 50-60 nucleotides 
and measures the area under the curve, one will see the relative 
percentages shown in the A+B column of Table II assuming that the 
amplification was equally efficient for the two sets of primers. If the 
amplifications were not equally efficient the values will change although 
this will not affect the ability to analyze the data unless the 
efficiencies were very different. For example, if A amplified 10 times as 
well as did B then marker B will be lost in the background and may not be 
seen. Such widely varying efficiencies are unexpected however. If neither 
A nor B is associated with the disease being studied in the affecteds, 
then the red spectrum shows the same relative percentages for each size 
band as for the normals. Note that this is true irrespective of the 
efficiencies of the amplifications. The one factor which will affect such 
a result will be the chance variation in the percentage of individuals 
with each size of marker. For example, assuming no association between the 
marker and the disease, it is expected that 50% of DNA samples from 
affected and unaffected persons will have an A marker of 56 nucleotides. 
If purely by chance only 30% of the affecteds had an A marker of 56 
nucleotides then the difference spectrum will not result in a zero signal 
but will give a peak. The larger the number of samples which can be 
pooled, the smaller will be the chance of this purely random false 
positive result. The pooling of DNA samples from many individuals causes 
such peaks to diminish in size and to be lost in the background or to be 
recognized as purely random differences in the two populations. 
If marker A is in fact associated with the disease and those with the 
disease all have an amplified marker A size of 54 nucleotides (assuming 
they have all received the mutated gene from a common ancestor and there 
has been no crossing over), there will be a dramatic difference spectrum 
seen. Assuming no association of marker B with the disease, the expected 
relative amount of each band in the affecteds is shown in Table III. 
TABLE III 
______________________________________ 
Expected % of Each Product in Affecteds for Which Marker A is 
Associated with the Disease and Marker B is not Associated 
with the Disease 
Size of Amplified 
Product % of Product for A 
% of Product for B 
% A + B 
______________________________________ 
50 0 10 5 
52 0 10 5 
54 100 30 65 
56 0 25 12.5 
58 0 10 5 
60 0 15 7.5 
______________________________________ 
Calculating the difference spectrum of the results in Tables II and III 
using the data in the A+B column, one gets the results shown in Table IV. 
TABLE IV 
______________________________________ 
Difference Spectrum 
Fragment Normal Affected Difference 
______________________________________ 
50 7.5 5 2.5 
52 10.0 5 5.0 
54 25.0 65 -40 
56 37.5 12.5 20 
58 7.5 5 2.5 
60 12.5 7.5 5.0 
______________________________________ 
As seen in Table IV, the difference spectrum is dramatic. This difference 
is seen despite the fact that there was overlap in the experiment with a 
marker which is not associated with the disease. From such data it becomes 
obvious that either marker A or marker B is associated with the disease. 
It will be necessary to repeat the experiment using markers A and B 
separately to determine which is the one associated with the disease. 
Although it is necessary to repeat the experiment with the markers 
individually, in practice several hundred markers may be examined with 
none of them showing an association. In such an instance there will be no 
need to repeat the experiments using the markers individually. It is 
necessary to do so only in the rare instances in which a marker is 
actually found. 
The example just given concerning markers A and B is for the relatively 
simple case in which only two markers overlap. As can be easily imagined 
or seen from the example shown in Table I, several markers could produce 
fragments which all overlap. For example, markers C, D, E and F will all 
produce amplified bands of 70 nucleotides. Nevertheless, a difference 
spectrum can still be observed although it will not be as dramatic as when 
only 1 or 2 markers are giving bands in a specific region. As noted 
earlier, it is best to use a mix of markers and primers which will give a 
wide spread to minimize the overlap. Adjusting the primer length can also 
be quite useful in spreading the bands. One trick along this line is to 
use primers which can shift the size of the amplified DNA fragment by a 
single base. In the examples above, it was stated that every primer was 15 
nucleotides in length. The repeat involved was the simple dinucleotide CA. 
This results in products which all have an even number of base pairs. 
Instead of using primers only 15 nucleotides in length, for half of the 
markers use one primer which is 15 nucleotides in length and use as the 
other primer one which is 16 nucleotides in length. For these markers the 
resulting amplified products will all be of an odd number of base pairs 
and can easily be distinguished from those of an even number of base 
pairs. This trick doubles the number of markers which can effectively be 
used at a single time. 
Many simple variations of the above methods can be easily imagined and 
these are considered to be within the scope of the present invention. Such 
variations include the labels used on the primers. A variety of different 
fluorescent colors may be chosen. One can even use radioactively labeled 
probes with two different radionuclides or use other nonfluorescent 
markers although such a method is much less convenient. Markers other than 
microsatellite regions of (CA).sub.n repeats can of course be used. The 
number of markers used at one time and the number of samples of DNA which 
are pooled can be varied dramatically. The number of markers which are 
pooled must be balanced between two competing practicalities, one being 
that the more markers which are used together increases the rate of 
screening of markers but which must be balanced against the fact that as 
more markers are used and increasingly overlap the smaller will be the 
signal of a true positive result. In practice it is useful to use between 
10-100 markers at a single time. The number of DNA samples which are 
pooled is important. The greater the number of samples used the better the 
averaging effect for the normals and the greater the summing effect for 
the affecteds.