bluuebunny/crossref_metadata_2025 huggingface dataset:
Construction of Small‐Insert Libraries Enriched for Short Tandem Repeat Sequences by Marker Selection
Authors: Pulido Jacqueline C. , Duyk Geoffrey M.
Published: June 2004

These protocols construct a representative small‐insert genomic DNA library in a phagemid vector. First, size‐selected DNA fragments are ligated into a phagemid vector. In the second protocol, the resulting small‐insert phagemid library is propagated in a bacterial strain combining mutations at the dut and ung loci, which permit incorporation of uracil in place of thymidine during DNA replication. Infection of the phagemid library with M13 helper phage permits recovery of this library as single‐stranded DNA (ssDNA). Finally, this uracil‐substituted ssDNA is used as a template for primer extension using an oligonucleotide whose sequence corresponds to the STR class of interest [e.g., (GATA)10] as primer. The products of this primer‐extension reaction are transformed into an E. coli strain maintaining wild‐type genes at the dut and ung loci. Under these conditions, uracil‐substituted ssDNA will be restricted from growing by the host‐encoded uracil‐N‐glycosylase, while the primer‐extended products are capable of replicating.
Colony Hybridization to Screen for Microsatellites
Authors: Hudson Thomas J.
Published: June 2004

Simple sequence length polymorphisms (SSLPs) for use as genetic markers are derived from the microsatellite repeat sequences found abundantly throughout eukaryotic genomes. Microsatellite DNA is identified in libraries of cloned DNA by colony hybridization to oligonucleotide probes containing simple sequence repeat (SSR) DNA, a common procedure in molecular biology laboratories. Plasmid or phagemid colonies are transferred to a nylon filter, grown briefly, fixed, and hybridized with one or more 32P‐labeled oligonucleotide probes. This protocol is adapted for the rapid processing of many filters at once and optimized for identification of long stretches of uninterrupted repetitive DNA sequences.
Characterization of (CA)n Microsatellite Repeats from Large‐Insert Clones
Authors: Litt Mike , Browne David
Published: June 2004

The most laborious part of developing (CA)n microsatellite repeats as genetic markers is constructing DNA clones to permit determination of sequences flanking the microsatellites. When cosmids or large‐insert phage clones are used as primary sources of (CA)n repeat markers, they have traditionally been subcloned into plasmid vectors such as pUC18 or M13 mp18/19 cloning vectors to obtain fragments of suitable size for DNA sequencing. This unit presents an alternative approach whereby a set of degenerate sequencing primers that anneal directly to (CA)n microsatellites can be used to determine sequences that are inaccessible with vector‐derived primers. Because the primers anneal to the repeat and not to the vector, they can be used with subclones containing inserts of several kilobases and should, in theory, always give sequence in the regions directly flanking the repeat. Degeneracy at the 3 end of each of these primers prevents elongation of primers that have annealed out‐of‐register.The most laborious part of developing (CA)n microsatellite repeats as genetic markers is constructing DNA clones to permit.
Genotyping by Ligation Assays
Authors: Nickerson Deborah A. , Ankener Wendy , Delahunty Claire , Kwok Pui‐Yan
Published: June 2004

This unit describes two methods of genotyping DNA sequences containing known nucleotide variations. The first protocol describes a colorimetric method for genotyping DNA samples amplified by the polymerase chain reaction (PCR) using an oligonucleotide ligation assay (OLA). The second protocol describes the ligase chain reaction (LCR), a method for simultaneously amplifying and genotyping genomic DNA samples. A describes the preparation of modified biotin‐ and digoxigenin‐labeled oligonucleotide primers.
High‐Throughput Genotyping Using the TaqMan Assay
Authors: Ranade Koustubh
Published: June 2004

This unit describes the use of the 5‐nuclease allelic discrimination assay, or TaqMan, for genotyping with single nucleotide polymorphisms (SNPs).

In this method, the region flanking the polymorphism, typically 100 to 150 bp, is amplified in the presence of two probes, each specific for one or the other allele. Probes have a fluor called “reporter” at the 5 end but do not fluoresce when free in solution because they have a “quencher“ at the 3 end that absorbs fluorescence from the reporter. The presence of two probes allows one to detect both alleles in a single tube. Moreover, because probes are included in the PCR, genotypes are determined without any post‐PCR processing, a feature that is unavailable with most other genotyping methods. The design of probes and primers is described first, followed by PCR conditions, and finally methods for assigning genotypes in an automated fashion are discussed.
High‐Throughput Genotyping with Primer Extension Fluorescent Polarization Detection
Authors: Kwok Pui‐Yan
Published: June 2004

The primer extension assay with fluorescence polarization (FP) detection is a versatile assay for single nucleotide polymorphism (SNP) genotyping. With proper design, this homogeneous assay works under universal conditions. Fluorescence polarization occurs when a fluorescent dye is excited by plane‐polarized light and is detected if the fluorescent dye is part of a large molecule. In assays where small fluorescent molecules are turned into large fluorescent products, FP provides a simple way to determine if the reaction has occurred without the need for separation or purification of the reaction mixture. In the primer extension assay, DNA polymerase incorporates the complementary, allelic nucleotide onto the SNP primer designed to anneal to the target DNA one base upstream of the polymorphic site. When a fluorescently labeled nucleotide is incorporated, high FP is observed for that dye and the genotype of the DNA sample is determined.
Construction of Somatic Cell Hybrids
Authors: Jackson Cynthia L.
Published: June 2004

Somatic cell hybridization is the method of choice to separate a chromosome of interest from the full chromosome complement and obtain a permanent source of the chromosome. This unit begins with the choice of fusion techniques and selectable markers for hybrids containing a chromosome of interest. The first set of protocols outline the production of whole‐cell hybrids by fusion of two cell lines: a monolayer (adherent) recipient and a donor that may be adherent or grown in suspension. The second set of protocols outline the production of micronuclei containing a limited number of chromosomes, and enucleation of the micronuclei to form microcells for fusion with recipient cells. Support protocols describe the preparation and use of cloning cylinders to isolate colonies in tissue culture, subcloning of whole‐cell hybrid populations to isolate lines that have segregated additional chromosomes, purification of microcell preparations, and molecular and cytogenetic methods for characterizing.
Construction and Assay of Radiation Hybrids
Authors: Richard Charles W. , Washington Sarah S.
Published: June 2004

This unit describes the irradiation and fusion of cells to produce radiation hybrids and the harvesting and expansion of RH colonies. A support protocol describes amplification of human chromosome fragments by PCR and analysis of those fragments by agarose gel electrophoresis.
Chromosome Preparation from Cultured Peripheral Blood Cells
Authors: Bangs Charles D. , Donlon Timothy A.
Published: June 2004

Chromosome preparations currently provide the only direct view of the genome as a whole. Although molecular methods allow a more detailed analysis of specific regions of the genome, the study of genetics is not complete without an appreciation of the metaphase cell. The stimulated T cell system described in this unit is the most widely used means of obtaining large numbers of mitotic cells for genetic analyses. Synchronization of the cell cycle in culture is described, combined with direct inhibition of chromosome condensation, to yield longer high‐resolution prophase or prometaphase preparations. Such preparations are used for detailed analysis of microdeletions or subtle rearrangements, fine breakpoint analysis, and refined mapping. Microscope slide preparation of mitotic chromosomes from harvested cell culture suspensions is also explained in the support protocol.
Microscopy and Image Analysis
Authors: Difilippantonio Michael J. , Ried Thomas , McNamara George , Bieber Frederick R.
Published: June 2004

This unit provides an overview of light microscopy, including objectives, light sources, filters, film, and color photography for fluorescence microscopy and fluorescence in situ hybridization (FISH). Computerized image analysis systems currently used in clinical cytogenetics are also discussed. Scanning and transmission electron microscopy as well as confocal microscopy are not covered in this unit despite their usefulness as invaluable tools for contemporary studies of biological systems.
High‐Resolution FISH Analysis
Authors: Heng Henry H.Q. , Tsui Lap‐Chee , Windle Bradford , Parra Irma
Published: June 2004

Map order, orientation, and gap or overlap distance of closely linked DNA probes may be determined using fluorescent hybridization to decondensed DNA. The linear arrangement of released chromatin fibers not only simplifies the task of gene ordering, but also provides higher resolution with probes separated by greater distances than can be achieved in FISH with intact interphase nuclei. The first basic protocol of this unit describes an alkaline lysis procedure for generating free chromatin from cultured cells for FISH analysis. A support protocol describes an empirical approach to optimize conditions for preparation of free chromatin. An provides a method for producing free chromatin from cultured lymphocytes with drug treatment. The second basic protocol, high‐resolution FISH mapping with free chromatin, is a modification of the method used for FISH mapping of interphase nuclei.
Comparative Genomic Hybridization
Authors: DeVries Sandy , Gray Joe W. , Pinkel Daniel , Waldman Frederic M. , Sudar Damir
Published: June 2004

Comparative Genomic Hybridization (CGH) is a powerful molecular cytogenetic technique that permits assessment of DNA copy number on a genome‐wide scale. Of note, this methodology uses tumor DNA as a probe for fluorescence in situ hybridization (FISH) to normal metaphase chromosomes and does not require dividing cells from the tumor specimen. This unit provides protocols for CGH, for preparation of metaphase chromosomes, tumor and normal DNAs for FISH and for the microscopy and image analysis of CGH experiments.
Construction of YAC Libraries with Large Inserts
Authors: Foote Simon , Denny Christopher
Published: June 2004

The yeast artificial chromosome (YAC) cloning system makes it possible to clone large pieces of genomic DNA into yeast. Libraries have been made containing clones with inserts in the megabase‐pair range. The basic protocol in this unit describes preparation of YAC vectors and transformation of ligated DNA into yeast spheroplasts. A support protocol describes titration of Lyticase to make spheroplasts. Additional support protocols detail two methods for partial digestion of genomic DNA: EcoRI restriction endonuclease‐EcoRI methylase competition and the partial digestion of genomic DNA by use of limiting amounts of Mg2+, respectively.
Construction of Chromosome Jumping and Linking Libraries in E. coli
Authors: Drumm Mitchell L.
Published: June 2004

Chromosome jumping allows the use of one point on a chromosome as a starting point for exploring another potentially distant point on the same chromosome without cloning the intervening sequences as in chromosome walking. A linking library consists of genomic fragments that each contain a particular restriction site with a marker inserted into it. Because the sequences flanking the marker represent the termini of two contiguous genomic restriction fragments, the linking clones can be used, in conjunction with a specific jumping library made with the same restriction enzyme, to expedite jumping between consecutive restriction fragments. Separate protocols present strategies for constructing general and specific jumping libraries from total genomic DNA. An additional protocol details construction of a linking library from flow‐sorted chromosomes. Support protocols provide instructions for preparing genomic insert DNA, supF gene fragments, and Chromosome jumping allows the use of one point on a chromosome as a starting point for exploring another potentially distant.
Screening Large‐Insert Libraries by PCR
Authors: Chinault A.Craig , Sternberg Nat L.
Published: June 2004

This unit describes procedures for screening large‐insert libraries by multistep polymerase chain reaction (PCR) analysis of DNA samples from clone pools. In the basic protocol, PCR amplification and agarose gel electrophoresis are used to identify successively smaller pools of DNA or colonies that contain clones with the appropriate‐sized amplification product. In the last screening step, individual clones are identified. The first and second support protocols describe the preparation of DNA and yeast‐cell pools for screening, and the first alternate protocol describes the preparation of crude lysates suitable for PCR from individual clones or small‐scale pools.
Rescuing YAC‐Insert Ends as E. coli Plasmids
Authors: Hermanson Gary G. , Evans Glen A.
Published: June 2004

End clones from YACs (terminal fragments of the YAC‐insert DNA cloned into a plasmid vector) are essential ingredients for contig building using chromosome walking strategies, for fluorescent in situ hybridization experiments, and for generating repeat‐free probes for pulsed‐field gel electrophoresis or genetic linkage analysis. The describes a method for rescuing the CEN (centromere) ends of YACs constructed in the vector pYAC4. However, because this method relies on XhoI and SalI sites, which are relatively rare in the mammalian genome, it is not always possible to obtain a subclone in this manner. An alternate protocol presents a method utilizing integrative plasmid‐rescue vectors to facilitate the isolation of both ends of any YAC clone even in the absence of convenient restriction enzyme sites.
Deriving Probes From Large‐Insert Clones by PCR Methods
Authors: Albertsen Hans , Thliveris Andrew , Riley John H. , Munroe David J. , Watkins Paul , Basson Craig T.
Published: June 2004

This unit describes several polymerase chain reaction (PCR)‐based methods to obtain DNA fragments from clones with large inserts without prior knowledge of the insert DNA sequence. The protocols can be categorized into three groups: (1) methods to generate DNA fragments at random representing the entire length of the cloned insert, (2) methods to generate DNA fragments representing the extremities of an insert, and (3) methods to generate complex probes suitable for fluorescence in situ hybridization. Support protocols describe direct cloning of these PCR products and the isolation of total yeast DNA from yeast artificial chromosome (YAC) clones.
Introduction of Large Insert DNA into Mammalian Cells and Embryos
Authors: Reeves Roger H. , Cabin Deborah E. , Lamb Bruce
Published: June 2004

This unit provides a set of protocols for introducing large insert DNA into cultured mammalian cells and embryos. Two different methods, spheroplast fusion and lipofection, are described for effecting transfer of YACs or gel‐purified YAC DNA into cells. Additional protocols discuss preparing and transferring BACs into cells by lipofection and into embryos by microinjection.
Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries
Authors: Osoegawa Kazutoyo , de Jong Pieter J. , Frengen Eirik , Ioannou Panayiotis A.
Published: June 2004

This unit describes the construction of BAC and PAC libraries. Two vectors, pCYPAC2 and pPAC4 have been used for preparing PAC libraries, and a new BAC vector pBACe3.6 has been developed for construction of BAC libraries. A support protocol describes preparation of PAC or BAC vector DNA for cloning by digestion with BamHI or EcoRI, simultaneous dephosphorylation with alkaline phosphatase, and subsequent purification through pulsed‐field gel electrophoresis (PFGE). For the preparation of high‐molecular weight DNA for cloning, support protocols provide procedures for embedding total genomic DNA from lymphocytes or animal tissue cells, respectively, in InCert agarose. Another support protocol details the next steps for the genomic DNA: partial digestion with MboI or with a combination of EcoRI endonuclease and EcoRI methylase, and subsequent size fractionation by preparative PFGE. The final support protocol covers the isolation of BAC and PAC plasmid DNA for analyzing clones.
Selective Isolation of Mammalian Genes by TAR Cloning
Authors: Kouprina Natalay , Larionov Vladimir
Published: June 2004

This unit describes procedures for selective isolation of specific chromosomal regions and entire mammalian genes as circular yeast artificial chromosomes (YACs). The isolation is based on transformation‐associated recombination (TAR) in yeast between genomic DNA and a linearized vector containing targeting sequences homologous to a region of interest. The details preparation of highly competent yeast spheroplasts and transformation of the spheroplasts by genomic DNA along with a TAR vector. Support protocols describe preparation of chromosome‐sized genomic DNA in solid agarose plugs, identification of positive clone pools among primary yeast transformants using PCR, and a quick method for preparing PCR templates from lysed spheroplasts. PCR analysis of each clone in the positive pool is performed to identify individual clones containing the gene of interest. A final support protocol describes retrofitting TAR‐isolated YACs into bacterial artificial chromosomes (BACs).
Isolation of Exons from Cloned DNA by Exon Trapping
Authors: Nisson Paul E. , Watkins Paul C. , Krizman David B.
Published: June 2004

Exon trapping is an RNA polymerase chain reaction (PCR) method to clone expressed sequences or exons directly from mammalian genomic DNA. The basic protocol in this unit describes the method for trapping internal exons from cosmid clones and the second basic protocol describes trapping of 3 terminal exons. An describes 3 terminal exon trapping, which avoids subcloning of target DNA by ligating it to the vector for direct transfection. A describes a rapid cloning procedure using uracil DNA glycosylase.
Identifying Transcribed Sequences in Arrayed Bacteriophage or Cosmid Libraries
Authors: Hochgeschwender Ute , Brennan Miles B.
Published: June 2004

This unit describes methods for identifying bacteriophage or cosmid clones that contain sequences present in the mRNA of a cell line or tissue. In the , a radiolabeled cDNA probe is synthesized by reverse transcription of poly(A)+ RNA isolated from the cell line or tissue of interest. The probe is incubated with DNA‐cellulose to remove highly repetitive sequences before it is used for hybridization analysis of filters from a phage or cosmid genomic library. After identification of positive clones from the library screen, the same probe can be used to screen Southern blots of restriction enzyme‐digested DNA from the positive clones. Support protocols describe preparation and testing of DNA‐cellulose.
Searching the NCBI Databases Using Entrez
Authors: Murphy Juliane , Baxevanis Andreas D.
Published: June 2004

One of the most widely‐used interfaces for the retrieval of information from biological databases is the NCBI Entrez system. Entrez capitalizes on the fact that there are pre‐existing, logical relationships between the individual entries found in numerous public databases. The existence of such natural connections, mostly biological in nature, argued for the development of a method through which all the information about a particular biological entity could be found without having to sequentially visit and query disparate databases. Two Basic Protocols describe simple, text‐based searches, illustrating the types of information that can be retrieved through the Entrez system. An Alternate Protocol builds upon the first Basic Protocol, using additional, built‐in features of the Entrez system, as well as providing alternative ways to issue the initial query. The Support Protocol reviews how to save frequently‐issued queries.
Amplification of Sequences from Affected Individuals
Authors: Watkins Hugh C.
Published: June 2004

This unit describes methods for obtaining DNA sequences from an individual affected by a genetic disorder. Target sequences that may be present at very low copy number in patient samples are amplified by the polymerase chain reaction (PCR). The first describes harvesting of mRNA from peripheral lymphocytes, which can even be utilized for genes with tissue‐specific patterns of expression. This technique has the advantage of ease of access to appropriate patient samples and also may provide a more efficient means of screening coding sequences than would analysis of individual exons. An alternate protocol describes modifications in PCR conditions that facilitate mutation analysis and sequencing. When the genomic sequence for a given candidate gene is known, the second may be used to obtain appropriate sequences for analysis of individual exons or noncoding regions of interest.
Detection of Mutations by RNase Cleavage
Authors: Watkins Hugh C. , Goldrick Marianna
Published: June 2004

Ribonucleases can specifically recognize and cleave RNA at the site of sequence mismatches in RNA‐DNA or RNA‐RNA hybrids. The cleavage products are then characterized by gel electrophoresis. In this unit, a procedure is presented for RNase cleavage of 32P‐labeled riboprobes (transcribed from a cloned copy of the normal sequence) that have been annealed to amplified sequences of a candidate gene or cDNA obtained from affected individuals. A explains how to prepare riboprobes from a genomic or cDNA template obtained from a nonmutant individual. An alternate protocol describes cleavage of RNARNA hybrids using a nonisotopic RNase cleavage mutation assay. Sequential PCR and in vitro transcription steps generate sufficient quantities of duplex RNA targets so that the cleavage products can be detected on a gel by ethidium bromide staining. The unit also discusses the use of alternative ribonucleases for cleaving singlebase mismatches.
Mismatch Detection Using Heteroduplex Analysis
Authors: Børresen Anne‐Lise
Published: June 2004

This protocol describes a technique that can be used to identify point mutations or single‐base polymorphisms in heterozygous individuals. This technique takes advantage of the fact that heteroduplex molecules containing single‐base mismatches can be separated under particular conditions of gel electrophoresis from nearly identical molecules containing no mismatches. RNA or DNA from a potentially heterozygous individual is amplified by PCR, and the products are denatured and allowed to renature, forming heteroduplexes. Renatured PCR products are run on nondenaturing mutation‐detection‐enhancement polyacrylamide gels (HydroLink MDE gels). On this gel, hybrid molecules containing a mismatch migrate more slowly than their corresponding homoduplexes. This protocol describes the analysis of unlabeled PCR products; however, radiolabeled PCR products can also be analyzed by this method.
Single‐Strand Conformation Polymorphism Analysis Using Capillary Electrophoresis
Authors: Larsen Lars Allan , Christiansen Michael , Vuust Jens , Andersen Paal Skytt
Published: June 2004

Single‐strand conformation polymorphism (SSCP) is one of the most frequently used mutation detection methods. This unit describes a method of SSCP with automated analysis by capillary electrophoresis in order to increase the capacity and throughput. A protocol is provided for sample preparation. For a medium throughput laboratory, a single capillary instrument, as described in this unit, may be quite sufficient. In many cases, however, screening for mutations in large population groups requires a high throughput, and this is best obtained through the use of a multi‐capillary instrument, as discussed.
Preparation and Culture of Products of Conception and Other Solid Tissues for Chromosome Analysis
Authors: Warburton Dorothy
Published: June 2004

In clinical settings, chromosome studies are usually performed on solid tissue other than solid tumors for one of two reasons: the tissue biopsy is the only tissue available from the patient, or tissues other than the standard peripheral blood lymphocytes must be examined because of suspected mosaicism. The,, anddescribe a method for culturing tissue samples to be used for preparation of metaphase chromosomes and subsequent examination or for biochemical or DNA analysis. Thedescribes mechanical disruption of the specimen to provide free access of the cells to the culture surface, whereasdescribes a more efficient enzymatic disruption procedure.anddescribe methods of collecting tissue samples., which can sometimes be applied to placental tissue, involves use of mitotically active chorionic villus tissue to prepare metaphase chromosome spreads directly without the need for cell culture.
Diagnosis of Fanconi Anemia by Diepoxybutane Analysis
Authors: Auerbach Arleen D.
Published: June 2004

Fanconi anemia (FA) is an autosomal recessive syndrome characterized by progressive pancytopenia and a high risk of malignancies, particularly acute myelogenous leukemia (AML). Diepoxybutane (DEB) analysis is the preferred test for FA because other agents have higher rates of false‐positive and false‐negative results. in this unit applies the DEB test to rule out a diagnosis of FA using a peripheral blood sample from the patient. provides instructions for working with DEB. describes staining slides for chromosome‐breakage analysis that is performed on unbanded metaphase preparations. provides a detailed method for applying the DEB test to cultured fibroblasts that grow as a monolayer attached to the bottom of the flask. outlines methods for using the DEB test for prenatal diagnosis of FA, utilizing fetal cells obtained by chorionic villus sampling (CVS), amniocentesis, or fetal blood sampling.Fanconi anemia (FA) is an autosomal recessive syndrome characterized by progressive pancytopenia and a high risk of malignanci.
Preparation of Amniocytes for Interphase Fluorescence In Situ Hybridization (FISH)
Authors: Schwartz Stuart , Micale Mark A. , Becker Laurie
Published: June 2004

Although FISH has been used to clarify deletions or structural rearrangements, recent work has focused increasingly on its applications to interphase analysis. This unit describes preparation of uncultured amniotic fluid cells for FISH analysis. Cells are swollen, then slides are prepared using either a cytocentrifuge or standard methods. These are then fixed and permeabilized for subsequent FISH. An describes attachment of amniocytes to a glass or plastic surface followed by hypotonic swelling, fixation, and permeabilization for subsequent FISH. Interphase FISH analysis of amniotic fluid cells is also described.
Molecular Cytogenetic Analysis of Telomere Rearrangements
Authors: Lese Christa M. , Ledbetter David H.
Published: June 2004

Genomic imbalances involving the telomeric regions of human chromosomes, which contain the highest gene concentration in the genome, are proposed to have severe phenotypic consequences. For this reason, it is important to identify telomere rearrangements and assess their contribution to human pathology. This unit describes the structure and function of human telomeres and outlines several FISH‐based methodologies that can be employed to study these unique regions of human chromosomes.
Molecular Analysis of Fragile X Syndrome
Authors: Nolin Sarah L. , Dobkin Carl , Brown W. Ted
Published: June 2004

The gene responsible for fragile X syndrome, fragile X mental retardation‐1 (FMR1), contains an unstable repeat sequence of (CGG)n. Additionally, an upstream promoter region of the gene—a CpG island—is abnormally methylated in most affected individuals. Amplification of CGG repeats and abnormal methylation show a correlation with affected status. The first basic and alternate protocols in this unit outline the use of the polymerase chain reaction (PCR) to rapidly detect the size of the repeat amplification. The second basic protocol describes the assessment of fragile X syndrome by direct Southern blot hybridization of genomic DNA digested with a pair of restriction enzymes, one of which is methylation‐sensitive. Extent of amplification and level of methylation can be simultaneously detected. A combination of the two techniques can be used to characterize the genotypes of individual members in identified fragile X families.
Analysis of Trinucleotide Repeats in Myotonic Dystrophy
Authors: Surh Linda C. , Mahadevan Mani , Korneluk Robert G.
Published: June 2004

Myotonic dystrophy is a genetic disorder characterized in 99% of clinically diagnosed families by an unstable CTG repeat in the 3‐untranslated region of a gene encoding a serine‐threonine protein kinase. There is no one method to detect the entire range of expansion sizes possible in affected patients, so current diagnostic approaches rely on analyzing samples by hybridization of both polymerase chain reaction (PCR)‐amplified CTG repeats (CTG‐PCR) and genomic DNA. In this unit, the describes the analysis of PCR‐amplified repeats transferred to a nylon membrane by Southern blotting and hybridized to an alkaline phosphatase‐labeled probe. The first support protocol describes a vacuum blotting technique for rapid transfer of the PCR product to the nylon membrane and the second support protocol describes the use of a radiolabeled oligonucleotide probe for hybridization. Analysis of genomic DNA by similar hybridization techniques is outlined in the second basic protocol.Myotonic dystrophy is a genetic disorder characterized in 99% of clinically diagnosed families by an unstable CTG repeat Myotonic dystrophy is a genetic disorder characterized in 99% of clinically diagnosed families by an unstable CTG repeat
Detection of Nonrandom X Chromosome Inactivation
Authors: Thouin Melissa M. , Giron James M. , Hoffman Eric P.
Published: June 2004

This unit describes a PCR‐based assay for distinguishing between the two X chromosomes in female cells and assessing the percentage of cells having each parental X chromosome active. Methylation of CpG residues in gene promoters is a major mechanism of transcriptional silencing. In mammalian female cells, hypermethylation is the way in which one X chromosome is inactivated. The X‐inactivation assay described in the relies on methylation sensitivity. In this unit, the highly polymorphic and therefore typically heterozygous (CAG)n region of the 5 end of the coding region of the human androgen receptor gene (HUMARA), at Xq11.2, is used to distinguish and compare the methylation activity of the X chromosomes.
Amplification‐Refractory Mutation System (ARMS) Analysis of Point Mutations
Authors: Little Stephen
Published: June 2004

The amplification‐refractory mutation system (ARMS) is a simple method for detecting any mutation involving single base changes or small deletions. ARMS is based on the use of sequence‐specific PCR primers that allow amplification of test DNA only when the target allele is contained within the sample. Following an ARMS reaction the presence or absence of a PCR product is diagnostic for the presence or absence of the target allele. The protocols detailed here outline methods that can be used to analyze human genomic DNA for one or more mutations. The describes the development and application of an ARMS test for a single mutation; the extends this to multiplex ARMS for the analysis of two or more mutations. The describes a rapid DNA extraction method from blood or mouthwash samples that yields DNA compatible with the type of tests described.The amplification‐refractory mutation system (ARMS) is a simple method for detecting any mutation involving single base change The amplification‐refractory mutation system (ARMS) is a simple method for detecting any mutation involving single base change
Molecular Analysis of Oxidative Phosphorylation Diseases for Detection of Mitochondrial DNA Mutations
Authors: Shoffner John M.
Published: June 2004

Oxidative phosphorylation (OXPHOS) diseases are caused by inherited or spontaneously occurring mutations in the mitochondrial DNA (mtDNA) or the nuclear DNA. Mutations in the mtDNA can be classified into two groups, rearrangements and point mutations. This unit describes a method for detecting rearrangements of the mtDNA, which involves Southern blot hybridization. Another protocol detects mtDNA point mutations using restriction analysis of polymerase chain reaction (PCR) products. The Southern blot method requires an mtDNA‐specific probe.
Protein Truncation Test
Authors: Vossen Rolf , den Dunnen Johan T.
Published: June 2004

The protein truncation test detects mutations at the protein level that lead to premature translation termination. The method has evolved considerably since is original publication in this manual. This thoroughly revised unit describes what is now the preferred method for performing the protein truncation test. Transcription and translation are performed in separate reactions; during translation, biotin‐labeled or N‐terminally tagged proteins are synthesized. The translation products are detected on immunoblots via chemiluminescence. An using coupled in vitro transcription/translation and radiolabeled proteins is also presented.
Internet Resources in Medical Genetics
Authors: Pagon Roberta A. , Tarczy‐Hornoch Peter , Hanson Nancy
Published: June 2004

This unit presents an overview of the most commonly used web‐based information resources for (1) clinicians seeking to apply molecular genetic testing to patient care or to understand the molecular basis of inherited diseases (e.g., GeneTests, or OMIM), (2) consumers seeking support group information or scientific and management information (e.g., Genetic Alliance, or The Family Village), and (3) professional societies. This unit presents an overview of the most commonly used web‐based information resources for (1) clinicians seeking to apply. This unit presents an overview of the most commonly used web‐based information resources for (1) clinicians seeking to apply.
Genotyping of Apolipoprotein E: Comparative Evaluation of Different Protocols
Authors: Ingelsson Martin , Shin Youngah , Irizarry Michael C. , Hyman Bradley T. , Lilius Lena , Forsell Charlotte , Graff Caroline
Published: June 2004

Disease‐associated gene polymorphisms provide both scientific insight into pathophysiological mechanisms and clinical information regarding risk and progression. Of special interest is the ɛ4 allele of the apolipoprotein E gene, which has emerged as a substantial risk factor for late‐onset forms of Alzheimer disease and also influences the risk of cardiovascular disease. Genotyping of apolipoprotein E can be performed by several methods; presented here are a quality and cost‐benefit analysis of four different protocols on a cohort of 42 clinical samples is included in the unit. Each method resulted in genotyping with a high sensitivity and specificity. The newer microtiter‐plate‐based high‐throughput techniques, fluorescence polarization and SNaPshot analysis, were as reliable as the traditional techniques of restriction fragment length polymorphism analysis and reverse hybridization. The reverse hybridization method tends to be more cost‐ and time‐effective when the number of analyses is limited, although economy of scale favors fluorescence polarization or SNaPshot analysis in larger studies. The latter approaches also provide the flexibility to investigate other polymorphic disease markers.
Methylation‐Specific PCR
Authors: Herman James G. , Baylin Stephen B.
Published: June 2004

Methylationspecific PCR is a rapid method used to determine the methylation status of DNA. Not only does methylation affect the expression of genes in normal cells, but it is now known that disease processes such as cancer can result in abnormal DNA methylation patterns. Methylationspecific PCR can be used to investigate imprinted genes, to assess human tumors for clonality by studying genes inactivated on the X chromosome, and to examine abnormally methylated CpG islands in neoplasia.
Constructing Tissue Microarrays for Research Use
Authors: Schweizer Martina Storz , Schumacher Lela , Rubin Mark A.
Published: June 2004

Tissue microarrays (TMAs) were recently developed to facilitate tissue‐based research. TMAs can be used for any type of study where standard tissue slides have previously been used. However, there are numerous advantages in using TMAs. TMAs allow for screening of a large number of tissue samples under similar experimental conditions. They are also useful in that they conserve tissue and resources and can greatly multiply the number of experiments that can be performed with a limited amount of tissue samples. The basic protocol in this unit discusses how to prepare and cut tissue microarray slides. The resulting arrays may then be constructed manually or automatically, as described in the support protocols.
Genetic Analyses on DNA Microarrays
Authors: Stein Lincoln D.
Published: June 2004

A great stir has coursed through the genetics community with the advent in 1996 of “chip technologies,” a series of related techniques that allow DNA hybridization‐based studies to be performed with unprecedented speed and parallelism. All chip technologies have in common a microarray, a small glass chip approximately one square centimeter in area, to which nucleotide sequences are bound. Fluorescently labeled nucleic acids are hybridized to the microarray and imaged with a laser scanner or fluorescence microscope. This unit offers an overview of the two dominant technologies, cDNA microarrays and oligonucleotide chips. It concludes with a discussion regarding how well microarrays perform real‐world expression analysis.
Analysis of Expression Data: An Overview
Authors: Grewal Anoop , Lambert Peter , Stockton Jordan
Published: June 2004

Microarray‐based methods generate large amounts of data that must be stored, visualized, and analyzed appropriately. The unit discusses normalization of transcriptional profiling data, statistically acceptable ways of validating measurements, database issues, and analysis of normalized data. This unit concentrates on the software package GeneSpring.
Biosafety in Handling Gene Transfer Vectors
Authors: Braun Andrew
Published: June 2004

This unit is devoted to safety issues that must be considered when generating and working with vectors for gene therapy. A short description of the relevant regulatory bodies and mechanisms is followed by a general discussion of containment and safety procedures. This is followed by vector‐specific guidelines for nine commonly used vector systems, including adenovirus, vaccinia, HSV‐1, lentivirus, and non‐viral vectors. The unit also outlines the problems encountered in taking a promising vector to clinical trials.This unit is devoted to safety issues that must be considered when generating and working with vectors for gene therapy.
Preparation of Adenovirus‐Polylysine‐DNA Complexes
Authors: Cotten Matt , Baker Adam , Birnstiel Max L. , Zatloukal Kurt , Wagner Ernst
Published: June 2004

This unit describes preparation of adenovirus‐polylysine‐DNA complexes, which is useful for transfection of DNA into a variety of cell types. A DNA complex is prepared with biotinylated adenovirus and streptavidin‐polylysine, coupled to transferrin, and used to transfect cells. Several support protocols describe methods for adenovirus growth and purification, biotinylation, inactivation with psoralen, and quantitation of the adenovirus particles. Additional support protocols descibes preparation of streptavidin‐polylysine and transferrin‐polylysine, necessary for the basic procedure. The DNA used for transfection must be free of lipopolysaccharide (LPS), and two methods for removing LPS are described. A more direct polylysine‐virus linkage that is simple and requires no exotic reagents can be used for transfection. This protocol requires polylysine‐modified adenovirus, prepared as described. An alternate protocol describes transfecting cells with free virus and DNA condensed with a polycation.
Adenoviral Vectors
Authors: He Tong‐Chuan
Published: June 2004

Adenoviral vectors have been widely used as efficient gene delivery vehicles for gene therapy and vaccine development, as well as gene function studies. The recently developed AdEasy system is a simple and efficient method for rapid generation of recombinant adenoviruses. Unlike traditional adenoviral vectors, the viral backbone described here is supplied as a supercoiled plasmid rather than as a linear viral DNA, facilitating backbone amplification. The recombination step is performed in E. coli rather than in mammalian cells, taking advantage of the high efficiency of homologous recombination in bacteria. This unit includes the complete set of protocols needed to generate recombinant adenoviruses using the AdEasy system.
Liposome Vectors for In Vivo Gene Delivery
Authors: Whitemore Mark , Li Song , Huang Leaf
Published: June 2004

Gene therapy with nonviral, cationic liposomebased vectors has demonstrated promising results in a variety of in vivo models and some clinical trials. In contrast to viral vectors, cationic liposomebased vectors usually induce just mild inflammatory reactions, making them suitable for repeated injections in animals. This unit describes the preparation and delivery of two liposomebased vectorsGene therapy with nonviral, cationic liposomebased vectors has demonstrated promising results in a variety of in vivo models.
Development of Molecular Genetic Interventions for HIV Infection
Authors: Woffendin Clive , Ranga Udaykumar , Nabel Gary J.
Published: June 2004

This unit includes a set of protocols for the ex vivo transfer of genes into CD4+ T cells, to be used in the initial evaluation of genes protecting against HIV infection in gene therapy protocols. The describes isolating and expanding CD4+ T cells from the patient. The cells are then transduced by either retroviral transduction or particle‐mediated gene transfer and reinfused into the patient. To monitor the effectiveness of gene transfer, genomic DNA is prepared from the patient's cells. Detection of vector DNA by PCR analysis of the patient's genomic DNA following gene transfer is also described in detail.
Gene Delivery to the Airway
Authors: Duan Dongsheng , Zhang Yulong , Engelhardt John F.
Published: June 2004

This unit describes generation of and gene transfer to several commonly used airway models. Isolation and transduction of primary airway epithelial cells are first described. Next, the preparation of polarized airway epithelial monolayers is outlined. Transduction of these polarized cells is also described. Methods are presented for generation of human bronchial xenografts as well as both ex vivo and in vivo gene transfer to these xenografts. Finally, a method for in vivo gene delivery to the lungs of rodents is included. Methods for evaluating transgene expression are given in the support protocols.
Gene Delivery to the Liver
Authors: Kozarsky Karen
Published: June 2004

Viral gene transfer to the liver has proven extremely effective in animal models and is currently being evaluated in clinical trials for a variety of metabolic disorders. In rodents, a single tail vein injection of an adenoviral vector can transduce most hepatocytes in vivo. This provides a convenient model for assessing vector design as well as for evaluating the effects of specific transgenes in genetic mouse models of human disease. Protocols for optimized in vivo hepatic gene transfer are described.
Overview of Human Identity Testing and Forensic Genetics
Authors: Bieber Frederick R.
Published: June 2004

This unit discusses the development of the field of forensic genetics, touching on the quality control and legal issues that are central to this area. It closes with a discussion of some caveats for interpreting forensic DNA results.
Collecting and Handling Samples for Parentage and Forensics DNA‐Based Genetic Testing
Authors: Bing David H. , Bieber Frederick R.
Published: June 2004

The unit covers Variable Numbers of Tandem Repeats (VNTR)based paternity analysis as well as the newer methods relying on PCR to analyze sequencespecific polymorphisms and microsatellite regions. The discussion of data analysis and probability calculations has been expanded to address a number of special circumstances, such as the lack of sample from an alleged father, motherless cases, and more.
Molecular Analysis of Paternity
Authors: Sozer Amanda C. , Kelly Charles M. , Demers Daniel B.
Published: June 2004

The unit covers Variable Numbers of Tandem Repeats (VNTR)based paternity analysis as well as the newer methods relying on PCR to analyze sequencespecific polymorphisms and microsatellite regions. The discussion of data analysis and probability calculations has been expanded to address a number of special circumstances, such as the lack of sample from an alleged father, motherless cases, and more.
RFLP Analysis of Forensic DNA Samples with Single‐Locus VNTR Genetic Markers
Authors: Bing David H. , Bieber Frederick R.
Published: June 2004

This unit covers the many and varied methods for extracting DNA from such diverse specimens as blood, tissue, stamps and envelopes, and cigarette butts, among others. Modifications to the methods that allow the DNA to be used for either PCR or Southern blotbased analyses are also included.
Manual Methods for PCR‐Based Forensic DNA Analysis
Authors: Bing David H. , Bieber Fredrick R.
Published: June 2004

This unit provides validated PCR‐based methods to test for sequence polymorphisms and length polymorphisms. It includes a description of the reverse dot blot method for detecting sequence polymorphisms. The forensic PCR systems used to detect length polymorphisms are based on detection of different‐sized PCR products following electrophoresis in either native or denaturing polyacrylamide gels. The unit offers a description of the amplification and detection of the variable number of tandem repeat (VNTR) length polymorphism at the D1S80 locus. It also describes the multiplex amplification of three separate autosomal short tandem repeats (STRs) and the X‐ and Y‐linked amelogenin alleles. These PCR products are electrophoresed on a denaturing polyacrylamide gel. Support protocols for creating permanent records of silver‐stained gels, checking the quality of reagents, and interpreting PCR‐based tests are provided.
Molecular Analysis of the Human Mitochondrial DNA Control Region for Forensic Identity Testing
Authors: Holland Mitchell M. , Huffine Edwin F.
Published: June 2004

This unit on mitochondrial DNA (mtDNA) highlights methods used to perform mtDNA sequence analysis on highly degraded biological material. The focus is on applications to forensic casework, and a number of case examples are provided. However, the laboratory working with ancient DNA, or any basic research laboratory, will benefit from this information.
Use of Chicken Models for the Analysis of Human Disease
Authors: Dodgson Jerry B. , Romanov Michael N.
Published: June 2004

The chicken has been a valuable model organism for genetic analysis since early in the last century. This unit provides an overview of the primary strategic approaches through which the chicken is used as a model for human genetics and disease. These approaches are reviewed with key advantages and disadvantages noted for each. In addition, the chicken offers a model system for several human inherited diseases, both single gene‐based and complex traits. This unit briefly describes two such models as examples: autoimmune pathology causing Hashimoto's thyroiditis and sex‐linked dwarfism.