Chapter 062. Principles of Human Genetics (Part 9) potx

Principles of Human Genetics Part 9 Nucleic Acid Hybridization Nucleic acid hybridization is a fundamental principle in molecular biology that takes advantage of the fact that the tw

Chapter 062 Principles of

Human Genetics

(Part 9)

Nucleic Acid Hybridization

Nucleic 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 Blot

Southern blotting is used to analyze whether genes have been deleted or rearranged It is also used to detect restriction fragment length polymorphisms (RFLPs) Genomic DNA is digested with restriction endonucleases and separated

by gel electrophoresis Individual fragments can then be transferred to a membrane and detected after hybridization with specific radioactive DNA probes Because single 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 normal

from mutant genes

Northern Blot

Northern blots are used to analyze patterns and levels of gene expression in different tissues In a Northern blot, mRNA is separated on a gel and transferred to

a membrane, and specific transcripts are detected using radiolabeled DNA as a probe This technique has been largely supplanted by more sensitive and comprehensive methods such as reverse transcriptase (RT)–PCR and gene expression arrays on DNA chips (see below)

Microarray Technology

A comprehensive approach to genome-scale studies consists of

microarrays, or DNA chips These microarrays consist of thousands of synthetic

nucleic acid sequences aligned on thin glass or silicon surfaces Fluorescently

labeled test sample DNA or RNA is hybridized to the chip, and a computerized scanner detects sequence matches Microarrays allow the detection of variations in DNA sequence and are used for mutational analysis and genotyping Alternatively, the expression pattern of large numbers of mRNA transcripts can be determined

by hybridization of RNA samples to cDNA or genomic microarrays This method has tremendous potential in the era of functional genomics and permits comprehensive analyses of gene expression profiles As one example, microarrays can be used to develop genetic fingerprints of different types of malignancies, providing information useful for classification, pathophysiology, prognosis, and treatment

The Polymerase Chain Reaction

The PCR, introduced in 1985, has revolutionized the way DNA analyses are performed and has become a cornerstone of molecular biology and genetic analysis In essence, PCR provides a rapid way of amplifying specific DNA fragments in vitro Exquisite specificity is conferred by the use of PCR primers, which are designed for a given DNA sequence The geometric amplification of the DNA after multiple cycles yields remarkable sensitivity As a result, PCR can be used to amplify DNA from very small samples, including single cells These properties also allow DNA amplification from a variety of tissue sources including blood samples, biopsies, surgical or autopsy specimens, or cells from hair or saliva PCR can also be used to study mRNA In this case, the enzyme RT is first

used to convert the RNA to DNA, which can then be amplified by PCR This

procedure, commonly known as RT-PCR, is useful as a quantitative measure of

gene expression

PCR provides a key component of molecular diagnostics It provides a strategy for the rapid amplification of DNA (or mRNA) to search for mutations by

a wide array of techniques, including DNA sequencing PCR is also used for the amplification of highly polymorphic di- or trinucleotide repeat sequences or the genotyping of SNPs, which allow various polymorphic alleles to be traced in genetic linkage or association studies PCR is increasingly used to diagnose various microbial pathogens

DNA Sequencing

DNA sequencing is now an automated procedure Although many protocols exist, the most commonly used strategy currently uses the capillary electrophoresis-based Sanger method in which dideoxynucleotides are used to randomly terminate DNA polymerization at each of the four bases (A,G,T,C) After separating the array of terminated DNA fragments using high-resolution gel

or capillary electrophoresis, it is possible to deduce the DNA sequence by examining the progression of fragment lengths generated in each of the four nucleotide reactions The use of fluorescently labeled dideoxynucleotides allows automated detection of the different bases and direct computer analysis of the

DNA sequence (Fig 62-5) Significant efforts are underway to develop faster, more cost-effective DNA sequencing technologies These include the use of pyrosequencing chemistries; whole-genome sequencing using solid-phase sequencing; mass spectrometry; detection of fluorescently labeled bases in flow cytometry; direct reading of the DNA sequence by scanning, tunneling, or atomic force microscopy; and sequence analysis using DNA chips