bluuebunny/crossref_metadata_2025_split huggingface dataset:
Strategies for Genotype Generation
Authors: Gilbert John , Vance Jeffrey 
Published: June 2004

AbstractThis unit describes methods for genotyping in families and individuals. Systematic step‐by‐step approaches are presented for setting up a study and carrying out the subsequent analyses. The discussion is divided into two parts. The Strategic Approach section provides an outline for the general approach to be used when beginning a new family or individual study, including guidelines for sample and data handling, cytogenetic analysis, choice of screening tests, and organization of DNA for genotyping. The commentary discusses some of these issues in more detail, providing information on molecular methods for genotyping, principally based on use of the polymerase chain reaction (PCR), in combination with simple sequence length polymorphisms (SSLPs) and, more recently, single nucleotide polymorphisms (SNPs). This discussion is followed by an outline of various genotyping techniques used in positional cloning and candidate gene analysis. Finally, potential new methods for automated mass genotyping are briefly discussed.
Analysis of Genetic Linkage Data for Mendelian Traits
Authors: Pericak‐Vance Margaret A. 
Published: June 2004

AbstractThis unit describes linkage analysis, an invaluable tool in mapping human disease genes. Linkage analysis is one of several methods used to map genes. Specifically, in linkage analysis cosegregation of two or more genes (traits) is examined in a family unit to determine if they segregate independently according to Mendel's laws or if they do not segregate independently because of their close physical proximity. The procedures outlined in this unit are not always straightforward, and a number of caveats are presented in the commentary regarding possible complications and confounding factors that may arise. Specific examples are given, but it is not possible to cover all possible scenarios or variables. The intention is to enable the reader to master basic principles used in the linkage approach and thus be in a position to undertake, with proper consultation, a linkage‐analysis study. This unit deals specifically with lod score analysis in Mendelian disorders.
Single‐Sperm Typing
Authors: Lien Sigbjorn , Szyda Joanna , Leeflang Esther P. , Hubert Rene , Zhang Lin , Schmitt Karin , Arnheim Norman 
Published: June 2004

AbstractThis unit presents protocols for sperm isolation using two different methods, amplification of simple sequence‐length polymorphisms (SSLP) and/or single nucleotide polymorphisms (SNP) from single cells or whole genome‐amplified single cells using primer extension preamplification (PEP), and discusses the statistical analysis of sperm‐typing recombination data. Newer methods for studying recombination over very short distances (a few kilobases) using total sperm DNA and allele‐specific PCR are also discussed.
PCR Methods of Genotyping
Authors: Hudson Thomas J. , Clark Chris D. , Gschwend Michele , Justice‐Higgins Erica 
Published: June 2004

AbstractTwo protocols discuss labeling PCR products with radioactive labels. The PCR products are then analyzed on a denaturing polyacrylamide gel and visualized by direct autoradiography. The resulting band pattern is used to define the SSLP genotype of the individual. Another method of genotyping uses a simple silver staining technique to detect PCR‐amplified SSLPs electrophoresed in denaturing polyacrylamide gels. In a fourth protocol, multiple unlabeled PCR products from one individual are pooled, separated on a denaturing polyacrylamide gel, transferred to a nylon membrane, and sequentially hybridized to nonradioactive end‐labeled probes derived from the primers used to amplify each SSLP marker. The probes are detected on film using chemiluminescence. Support protocols describe the preparation of an M13 sequence ladder size standard and digoxigenin labeling of the probe, both of which are required for the chemiluminescent method.
Single‐Nucleotide Polymorphism Genotyping Using Microarrays
Authors: Patil Nila , Nouri Nassim , McAllister Linda , Matsukaki Hajime , Ryder Thomas 
Published: June 2004

AbstractThis unit provides a set of protocols for high throughput SNP genotyping using oligonucleotide microarrays. The basic protocol utilizes allele‐specific hybridization in which the microarray provides both the allele discrimination and read‐out. In the alternate approach, the allele discrimination is accomplished in solution and then the array is used to provide the read‐out of the genotyping reaction.Single nucleotide polymorphisms (SNPs) offer several advantages over other genetic markers including their high density in the.
In Situ Hybridization to Metaphase Chromosomes and Interphase Nuclei
Authors: Knoll Joan H. M. , Lichter Peter 
Published: June 2004

AbstractIn situ hybridization is used to determine the chromosomal map location and the relative order of genes and DNA sequences within a chromosomal band. It can also be used to detect aneuploidy, gene amplification, and subtle chromosomal rearrangements. Fluorescence in situ hybridization (FISH), probably the most widely used method, is described in the first basic protocol. Two support protocols are provided to amplify weak fluorescent signals obtained in FISH. Nonisotopic probes can also be detected by enzymatic reactions using horseradish peroxidase or alkaline phosphatase, as described in alternate protocols. Nonisotopic labeling of DNA probes by nick translation is described in a support protocol. The order of closely spaced FISH probes along chromosomes in interphase nuclei can be determined. A basic protocol for isotopic in situ hybridization (IISH) with 3H is provided followed by a support protocol for preparation of autoradiographic emulsion.
Chromosome Microdissection
Authors: Meltzer Paul , Bittner Michael 
Published: June 2004

AbstractThis unit describes chromosome microdissection methodologies that are useful in both clinical and research laboratories. In combination with FISH, region‐specific probes can be made to determine the origin of marker chromosomes. Another application of the microdissection technology is in the generation of regionspecific cDNA libraries, known as microdissection‐mediated cDNA capture.
Mitotic Chromosome Preparations from Mouse Cells for Karyotyping
Authors: Akeson Ellen C. , Davisson Muriel T. 
Published: June 2004

AbstractThis unit contains protocols for the preparation of mitotic chromosomes from mouse peripheral blood, Giemsa banding of those chromosomes, and classification into a karyotype, including recognition of some common pitfalls of misidentification and information for determining aberrant chromosomes. The methods described can be used to identify visible chromosomal rearrangements and their precise cytological breakpoints in the living mouse. In conjunction with fluorescent in situ hybridization (FISH), the metaphase spreads can also be used for the linear placement of loci on a chromosome and for determining the insertion site(s) of a foreign transgene.
Single‐Nucleotide Sequence Discrimination In Situ Using Padlock Probes
Authors: Nilsson Mats , Landegren Ulf , Antson Dan‐Oscar 
Published: June 2004

AbstractDNA ligases are very sensitive to mismatches at the DNA ends to be joined through ligation. This mechanism has been exploited to distinguish DNA sequence variants in situ using so‐called padlock probes. Padlock probes are linear oligonucleotides with target‐complementary sequences at both ends, and an on‐target‐complementary segment in between. The end sequences are brought next to each other upon hybridization to the target DNA sequence, and if the ends are perfectly matched to the target sequence, they can be joined by a DNA ligase. Padlock probes detect target sequences with very high specificity, because both probe segments must hybridize to the target for circularization to occur. This unit presents a protocol for discrimination between closely similar DNA sequences in situ using padlock probes. A discussion of methods for greatly amplifying the signal from circularized probes is also included.DNA ligases are very sensitive to mismatches at the DNA ends to be joined through ligation.
Strategies for Large‐Insert Cloning and Analysis
Authors: Moir Donald T. , Smith Douglas R. 
Published: June 2004

AbstractThis revised introduction provides an overview of several new methods and resources available to researchers involved in generating and screening large insert clones. Emphasis has been placed on the use of bacterial artificial chromosome clones. The table of available libraries has been updated and focused on the proven, widelyused libraries of YAC and BAC clones. Finally, the table of physical mapping databases has been expanded to include web sites for each of the individual human chromosomes, where available, as well as the wholegenome efforts with radiation hybrid and STS/EST mapping. A rapid convenient degenerate primervector primer method for obtaining PCR fragments representing the ends of P1 clone inserts has been added to Unit here. In addition, Unit here has been expanded to include a method for building BAC contigs by sequencing BAC insert ends.This revised introduction provides an overview of several new methods and resources available to researchers involved in gener This revised introduction provides an overview of several new methods and resources available to researchers involved in gener
Construction of Bacteriophage P1 Libraries with Large Inserts
Authors: Sternberg Nat , Shepherd Nancy S. 
Published: June 2004

AbstractThe bacteriophage P1 cloning system was originally developed as an alternative to YAC and cosmid systems for cloning high‐molecular‐weight genomic DNA. This unit details the preparation of the bacteriophage P1 library. Three support protocols provide the raw materials for the basic procedure, including the vector (pAd10sacBII), the mammalian DNA inserts, and the two packaging extracts that contain the viral proteins necessary to construct a P1 bacteriophage incorporating the vector and insert. A fourth support protocol describes how to induce replication of the plasmids cloned in the basic protocol, isolate the cloned DNA, and analyze the final products.
Screening Large‐Insert Libraries by Hybridization
Authors: Ross (gridding: YAC, BAC, and PAC colony hybridization) Mark T. , LaBrie (BAC/PAC colony hybridization and analysis) Sam , McPherson (overgo probes and hybridization) John , Stanton Vincent P. 
Published: June 2004

AbstractThree types of hybridization screening are discussed in this unit colony hybridization, DNA hybridization, and YAC Alu‐PCR‐product hybridization. The first protocol describes methods for arraying large‐insert clones. Subsequent protocols discuss hybridizing YAC and BAC/PAC colony blots. An alternate protocol provides a guide for preparing and hybridizing with, high‐specific‐activity oligonucleotide probes called “overgo” probes. Support protocols indicate the procedures for identifying the plate and well address of positive clones by manual and software‐assisted methods. Hybridization of Alu‐PCR‐product probes to Alu‐PCR products of YAC clones are provided. An alternate protocol describes a method for preparing DNA from individual YACs and for producing and using Alu‐PCR product blots from individual YACs. Additional support protocols provide guidelines for preparing Alu‐PCR probes from cosmids, YACs, BACs/PACs, and somatic cell hybrids, and describe the use of 96‐well plates to radiolabel and spin‐column‐purify large numbers of probes. A method for YAC walking is also provided.Three types of hybridization screening are discussed in this unit colony hybridization, DNA hybridization, and YAC Alu‐PCR.
Purification and Characterization of YACs Containing Large Inserts
Authors: Gemmill Robert M. , Bolin Richard , Strauss William M. , Pavan William 
Published: June 2004

AbstractThis unit provides protocols for characterizing DNA segments cloned in YACs and for purifying YACs from yeast chromosomes. The first basic protocol describes Southern blotting and partial‐digest restriction analysis of YACs. These methods are useful for determining the size and complexity of the cloned insert DNA, the presence and location of particular restriction sites or sequences, and even the species of origin of the insert DNA (indicated by hybridization to species‐specific repetitive elements such as Alu repeats). The second basic protocol describes gel purification of YACs for use in procedures requiring pure YAC DNA, such as mammalian‐cell transformation and subcloning into smaller insert vectors. The third basic protocol details characterizing and analyzing YACs: in vivo fragmentation via homologous recombination with specialized fragmentation vectors containing specific probe sequences or repetitive elements, followed by Southern blotting with YAC‐ and human‐derived probes.
Constructing Contigs from Large‐Insert Clones
Authors: Foote Simon , Marshall Vikki , Munroe David J. , Segre Julia A. 
Published: June 2004

AbstractThis unit describes three approaches that are widely used to define alignments between overlapping clones bearing large‐insert genomic DNA and to generate extensive contiguous overlapping sets of clones (contigs). The three approaches are sequence‐tagged site (STS) content mapping, repetitive‐element hybridization fingerprinting, and Alu‐PCR fingerprinting. Methods for isolating the necessary BAC DNA suitable for automated fluorescent sequencing and generating new STS markers are discussed in support protocols. An alternate protocol presents repetitive‐element hybridization fingerprinting to detect overlaps and build contigs with full‐genomic YAC libraries.
Building Larger YACs by Recombination
Authors: Silverman Gary A. 
Published: June 2004

AbstractDespite the relatively large cloning capacity of YACs, many genomic regions or individual genes are not cloned intact, but are represented as a collection of overlapping clones or contigs. Fortunately, the relatively high frequency and fidelity of homologous recombination in Saccharomyces cerevisiae can be used to reconstruct intact genes within a single clone by splicing together overlapping DNA segments. This unit describes two protocols for carrying out such homologous recombination; one relies on the meiotic phase of the yeast cycle, while the other utilizes the mitotic phase of the yeast life cycle.Despite the relatively large cloning capacity of YACs, many genomic regions or individual genes are not cloned intact.
Transfer of YAC Clones to New Hosts by Karyogamy‐Deficient Mating
Authors: Spencer Forrest , Simchen Giora 
Published: June 2004

AbstractThis unit provides a protocol for moving yeast artificial chromosome (YAC) clones to new yeast hosts using basic microbial techniques and pulsed‐field gel analysis. In contrast to other methods that can be used to transfer YAC clones, this requires neither optimization to achieve high‐efficiency DNA‐mediated transformation of chromosome‐sized DNA nor specialized equipment for tetrad dissection and analysis. Instead, chromosome (YAC) transfer is selected in rare segregants (“YACductants”) from a yeast mating that is rendered incomplete in most cell pairings by the presence of a kar1 (karyogamy‐deficient) mutation in either parental strain. The in this unit details the transfer of a YAC clone from yeast strain AB1380 (host to nearly all existing YAC libraries) to YPH925, a strain with nonreverting genetic markers compatible with existing plasmid constructs useful in YAC modification.
Navigating Public Physical Mapping Databases
Authors: Stein Lincoln D. 
Published: June 2004

AbstractThis unit provides concise overviews of the many physical mapping resources available and relates them to the genetic and transcript maps. Useful information on resolution of the maps, how to access them, and how to interpret them is compiled and presented in a clear fashion. Especially useful is a set of detailed protocols describing how to construct an STS marker and how to map it by means of available yeast artificial chromosomes (YACs). An additional protocol describes accessing EST marker maps.
Direct Selection of cDNAs Using Genomic Contigs
Authors: Lovett Michael 
Published: June 2004

AbstractIn the , uncloned cDNA or cDNA from a library with highly repetitive sequences suppressed are hybridized to biotinylated genomic DNAs. The selected sequences are amplified by PCR and rehybridized to create a secondary‐selected enriched cDNA library. Biotinylation of cloned genomic DNA is presented in the first support protocol. Linker addition and suppression of repeats in uncloned DNA is presented in the second support protocol and insert amplification and suppression of repeats from libraries is detailed in the third support protocol.In the , uncloned cDNA or cDNA from a library with highly repetitive sequences suppressed are hybridized to biot.
Identification of Intron/Exon Boundaries in Genomic DNA by Inverse PCR
Authors: Albertsen Hans , Thliveris Andrew 
Published: June 2004

AbstractThis unit describes identifying intron/exon boundaries in genomic DNA by comparing nucleotide sequences of genomic DNA to cDNA. Cloned genomic DNA is prepared for inverse polymerase chain reaction (PCR) by digesting the DNA with a restriction enzyme and circularizing the restriction fragments by ligation. Diverging primer pairs for each exon are designed on the basis of the cDNA sequence. The circularized restriction fragments are amplified using these diverging primers, the PCR product is sequenced, and the sequence is compared to the cDNA sequence to determine the location of the intron/exon boundaries. The lower complexity of cloned DNA (e.g., YAC, P1, or cosmid DNA) facilitates preparation of good template.This unit describes identifying intron/exon boundaries in genomic DNA by comparing nucleotide sequences of genomic DNA to cDNA.
GrailEXP and Genome Analysis Pipeline for Genome Annotation
Authors: Uberbacher Edward C. , Hyatt Doug , Shah Manesh 
Published: June 2004

AbstractThe Gene Recognition and Analysis Internet Link (GRAIL) is one of the most widely used systems for evaluating the protein‐coding potential of anonymous DNA sequences. This unit describes the use of the XGRAIL and genQuest client‐server applications to locate exons in DNA sequences, to develop gene models, and to search databases for homologs. A support protocol describes how to obtain the GRAIL and genQuest client software by anonymous FTP.
Detection of Mutations by Single‐Strand Conformation Polymorphism (SSCP) Analysis and SSCP‐Hybrid Methods
Authors: Warren William , Hovig Eivind , Smith‐Sørensen Birgitte , Børresen Anne‐Lise , Fujimura Frank K. , Liu Qiang , Feng Jinong , Sommer Steve S. 
Published: June 2004

AbstractSingle‐strand conformation polymorphism (SSCP) analysis detects mutations based on the fact that single‐nucleotide changes in DNA sequences alter the mobility of single‐stranded DNA in nondenaturing gels. Four methods for detecting mutations based on SSCP are described here. (1) Traditional SSCP analysis is technically easy and can be used for screening large numbers of samples. SSCP‐hybrid methods detect mutations based on either an SSCP effect or an altered component independent of the SSCP effect. (2) Dideoxy fingerprinting (ddF) involves PCR amplification of the target and creation of a set of dideoxy‐terminated strands with the mutation. (3) Bi‐directional dideoxy fingerprinting (Bi‐ddF) involves production of two sets of dideoxy‐terminated strands that are generated from two different primers. (4) Restriction endonuclease fingerprinting (REF) involves cleavage of the amplified target with five to six groups of restriction endonucleases.
Chemical Cleavage of Heteroduplex DNA to Identify Mutations
Authors: Cotton Richard G. H. , Grompe Markus 
Published: June 2004

AbstractMutation screening by the chemical‐cleavage method is based on the fact that mismatched cytosine (C) and thymidine (T) are more reactive with the compounds hydroxylamine and osmium tetroxide, respectively, than are Watson and Crick‐paired cytosine and thymidine bases. In this protocol, an excess of unlabeled target DNA is hybridized with labeled wild‐type DNA probe and heteroduplexes are formed. One aliquot is treated with hydroxylamine, which reacts with mismatched C bases. Another aliquot is treated with osmium tetroxide, which reacts with mismatched T bases. The reactions are mixed with piperidine; the strands are then cleaved at the sites where hydroxylamine and osmium tetroxide react. Cleaved fragments are then electrophoresed and sized on polyacrylamide gels, identifying the point of cleavage (and hence the position of the mutation). Then only a small portion of the mutant gene needs to be sequenced to define a single change between two DNA sequences.
Mutation Detection by Cycle Sequencing
Authors: Thierfelder Ludwig 
Published: June 2004

AbstractCandidate genes are screened for mutations by a DNA sequencing procedure known as cycle sequencing. First, a segment of the candidate gene is PCR amplified from the genomic DNA of an affected individual. The PCR product is then subjected to multiple rounds of further amplification in a thermal cycler using a heat‐stable DNA polymerase in the presence of different dideoxynucleotides and a radiolabeled primer. The resulting 32P‐labeled sequence reaction products are fractionated on a denaturing polyacrylamide gel and visualized by autoradiography. DNA segments on the order of 200 bp from 10 to 30 individuals can be screened on each gel. Cycle sequencing eliminates the need to subclone genomic fragments or PCR products, which makes it a much simpler method than conventional sequencing for identifying mutations.
Heterozygote Detection Using Automated Fluorescence‐Based Sequencing
Authors: Dabora Sandra L. , Arad Michael , Barr Scott , Kim Jae Bum 
Published: June 2004

AbstractPoint mutations are the most common type of polymorphism. These mutations can occur in one allele, while the other allele retains the normal sequence; detecting such sequence variants in a heterozygous individual's DNA, carrying both the normal allele and the mutant allele, can be difficult. In this unit, a strategy is presented for detecting such sequence variation using automated fluorescence‐based sequencing traces, and analysis with software tools that assist with the identification of sequence variation. A protocol is given that has been used to identify heterozygote mutations in TSC1 and TSC2, the causative genes for the autosomal dominant disorder tuberous sclerosis complex (TSC), as well as for a number of other inherited disorders in humans. Common genetic diseases like hypertrophic cardiomyopathy (HCM) are caused by autosomal dominant mutations in a subset of sarcomere proteins. An alternate protocol describes an approach for large‐scale sequencing adapted for serial screening for HCM mutations.
DNA Mutation Detection Using Denaturing High‐Performance Liquid Chromatography (DHPLC)
Authors: Oefner Peter J. , Underhill Peter A. 
Published: June 2004

AbstractThis method permits the rapid detection of singlebase substitutions and insertions or deletions of up to 1.5 kb in PCRamplified DNA fragments. The method exploits the differential retention of homo and heteroduplex DNA species under conditions of partial thermal denaturation and provides an efficient method to rapidly compare DNA fragments for sequence variation, once this variation has been identified by conventional sequencing.
Mutation Nomenclature
Authors: den Dunnen Johan T. , Antonarakis Stylianos E. 
Published: June 2004

AbstractAs part of the Human Genome Variation Society (formerly known as the HUGO Mutation Database Initiative), a committee was formed to suggest standards for the description of sequence variants in DNA, RNA, and protein sequences. The committee proposed that the nomenclature should be unequivocal, precise, and short, and should prevent any possible confusion and follow existing practice as much as possible. To “spread the word,” the nomenclature rules were published at regular intervals. This unit summarizes these nomenclature recommendations, which stimulated a uniform and unequivocal description of sequence variants in literature.