In 1975, Edwin Southern first combined the specificity of restriction endonu-clease digestion with gel electrophoresis to identify sequence variations in fragments of genomic DNA (Table 7.1).1_34 Restriction enzyme digestion generated a collection of controlled, predictable DNA fragments, and gel electrophoresis of DNA fragments immobilized on a membrane provided an efficient, economic means to array the collection of fragments by size. The simplicity and reliability of the method for molecular analysis quickly led to its adoption in many labo-ratories.1,2 In 1977 Fred Sanger devised a method for direct sequencing of ge-nomic DNA that used chain termination by dideoxy-nucleotides to specify the genetic change and define its genomic location.3 In the same year, Alwine developed the ''Northern blot'' version of Southern's technique,4 in which a complex RNA sample is size separated and then transferred to an immobilization membrane. This technique established the link between the genomic sequence and messenger RNA (mRNA) expression.
In 1980, Wyman and White showed that probing DNA fragments by restriction enzyme digestion and hybridization yielded altered electrophoretic banding patterns of diagnostic value called restriction fragment length polymorphisms (RFLPs).5 Wyman and White also reported the accidental discovery of repetitive hypervariable sequences throughout the genome. Because they exhibited variable numbers of repeats within a single locus and between loci, these sequences were termed hypervariable. Encouraged by this report, in 1985 Jeffreys developed the technique of ''DNA fingerprinting.''8,35 For this purpose, Jeffreys developed probes that hybridize to these hypervariable loci, which were also dubbed ''minisatellites'' or ''variable number tandem repeats'' (VNTRs), and yielded RFLPs arrayed in Southern blots. DNA fingerprinting has been routinely used for geno-mic mapping and also for individual identification in criminal investigations, paternity disputes, zygosity testing in twins, monitoring transplants, and wildlife forensics.35
Subsequently, ''dot'' blots and ''slot'' blots were developed.16,17 Dot blots'' and ''slot blots'' were also hybridization methodologies that take their name from the circular and slotted wells in templates used to present test samples to a membrane surface. Neither of these methods requires restriction digestion or electro-phoresis, and they are faster and easier for hybridization screening than Southern blotting, especially for samples too small or too damaged to undergo purification, digestion, and electrophoresis, or when many samples are to be screened simultaneously using different sets of hybridization probes. They are, however, somewhat less informative because they do not provide RFLP information and are more prone to nonspecific background hybridization. A few years later, after the invention of the polymerase chain reaction (PCR),12 the ''reverse dot blot'' was developed. The ''reverse dot blot'' makes use of probe collections instead of the DNA test samples immobilized on the membrane. DNA to be analyzed is submitted to PCR amplification, labeled with a detectable marker, and hybridized to the array of immobilized probes.19 With PCR, smaller and smaller amounts of DNA could be analyzed still more rapidly. Reverse dot blot probe sets could be
Table 7.1 Charting the Evolution of Genomics Tools
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