Chapter 062. Principles of Human Genetics (Part 9)

Nucleic Acid HybridizationNucleic acid hybridization is a fundamental principle in molecular biology that takes advantage of the fact that the two complementary strands of nucleic acids bind, or hybridize, to one another with very high specificity. The goal of hybridization is to detect specific nucleic acid (DNA or RNA) sequences in a complex background of other sequences. This technique is used for Southern blotting, Northern blotting, and for screening libraries (see above). Further adaptation of hybridization techniques has led to the development of microarray DNA chips.Southern BlotSouthern blotting is used to analyze whether genes have been deleted or rearranged....
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Chapter 062. Principles of Human Genetics (Part 9) Chapter 062. Principles of Human Genetics (Part 9) Nucleic Acid Hybridization Nucleic acid hybridization is a fundamental principle in molecular biologythat takes advantage of the fact that the two complementary strands of nucleicacids bind, or hybridize, to one another with very high specificity. The goal ofhybridization is to detect specific nucleic acid (DNA or RNA) sequences in acomplex background of other sequences. This technique is used for Southernblotting, Northern blotting, and for screening libraries (see above). Furtheradaptation of hybridization techniques has led to the development of microarrayDNA chips. Southern Blot Southern blotting is used to analyze whether genes have been deleted orrearranged. It is also used to detect restriction fragment length polymorphisms(RFLPs). Genomic DNA is digested with restriction endonucleases and separatedby gel electrophoresis. Individual fragments can then be transferred to a membraneand detected after hybridization with specific radioactive DNA probes. Becausesingle base-pair mismatches can disrupt the hybridization of short DNA probes(oligonucleotides), a variation of the Southern blot, termed oligonucleotide-specific hybridization (OSH), uses short oligonucleotides to distinguish normalfrom mutant genes. Northern Blot Northern blots are used to analyze patterns and levels of gene expression indifferent tissues. In a Northern blot, mRNA is separated on a gel and transferred toa membrane, and specific transcripts are detected using radiolabeled DNA as aprobe. This technique has been largely supplanted by more sensitive andcomprehensive methods such as reverse transcriptase (RT)–PCR and geneexpression arrays on DNA chips (see below). Microarray Technology A comprehensive approach to genome-scale studies consists ofmicroarrays, or DNA chips. These microarrays consist of thousands of syntheticnucleic acid sequences aligned on thin glass or silicon surfaces. Fluorescentlylabeled test sample DNA or RNA is hybridized to the chip, and a computerizedscanner detects sequence matches. Microarrays allow the detection of variations inDNA sequence and are used for mutational analysis and genotyping. Alternatively,the expression pattern of large numbers of mRNA transcripts can be determinedby hybridization of RNA samples to cDNA or genomic microarrays. This methodhas tremendous potential in the era of functional genomics and permitscomprehensive analyses of gene expression profiles. As one example, microarrayscan be used to develop genetic fingerprints of different types of malignancies,providing information useful for classification, pathophysiology, prognosis, andtreatment. The Polymerase Chain Reaction The PCR, introduced in 1985, has revolutionized the way DNA analysesare performed and has become a cornerstone of molecular biology and geneticanalysis. In essence, PCR provides a rapid way of amplifying specific DNAfragments in vitro. Exquisite specificity is conferred by the use of PCR primers,which are designed for a given DNA sequence. The geometric amplification of theDNA after multiple cycles yields remarkable sensitivity. As a result, PCR can beused to amplify DNA from very small samples, including single cells. Theseproperties also allow DNA amplification from a variety of tissue sources includingblood samples, biopsies, surgical or autopsy specimens, or cells from hair orsaliva. PCR can also be used to study mRNA. In this case, the enzyme RT is firstused to convert the RNA to DNA, which can then be amplified by PCR. Thisprocedure, commonly known as RT-PCR, is useful as a quantitative measure ofgene expression. PCR provides a key component of molecular diagnostics. It provides astrategy for the rapid amplification of DNA (or mRNA) to search for mutations bya wide array of techniques, including DNA sequencing. PCR is also used for theamplification of highly polymorphic di- or trinucleotide repeat sequences or thegenotyping of SNPs, which allow various polymorphic alleles to be traced ingenetic linkage or association studies. PCR is increasingly used to diagnosevarious microbial pathogens. DNA Sequencing DNA sequencing is now an automated procedure. Although many protocolsexist, the most commonly used strategy currently uses the capillaryelectrophoresis-based Sanger method in which dideoxynucleotides are used torandomly terminate DNA polymerization at each of the four bases (A,G,T,C).After separating the array of terminated DNA fragments using high-resolution gelor capillary electrophoresis, it is possible to deduce the DNA sequence byexamining the progression of fragment ...