Three important techniques have been developed for nucleic acid manipulation and analysis of genomic sequences.
The first of these is molecular cloning. In its simplest form, this involves first cutting or breaking the target nucleic acid, i.e. DNA, into smaller fragments (typically by restriction endonuclease digestion) and inserting the fragments into a biological vector. The assortment of DNA fragments is then maintained and amplified by the replication of the vector DNA in vivo. Separation of the copies of cloned DNA in this "library" is accomplished by dilution and subsequent growth of bacterial colonies or phage plaques from single organisms bearing copies of only one of the original DNA fragments. Identification of the clones of interest is done by hybridization of a specific labelled probe with the DNA released from each colony or plaque.
More recently, a second technique was developed called the Polymerase Chain Reaction or PCR. This technique can be used to isolate and amplify sequences of interest. The technique allows the definition of any "target" portion of a nucleic acid sequence by the sequences which lie adjacent to it. Consequently, hybridization of nucleic acid primers at these adjacent sites permits the replication of only the intervening target sequence and the adjacent primer sites. The selective amplification by repeated replication in this way results directly in the separation of the desired fragment (or subset of sequences) by effective dilution of all other unwanted sequences by replicated copies of the target sequence. Identification is then carried out by hybridization against a known probe, or more frequently, by simple size analysis by agarose or polyacrylamide gel electrophoresis to confirm that the desired target sequence has been amplified.
A third major technique used for comparative genomic analysis is called Restriction Fragment Length Polymorphism, or RFLP, analysis. Insertions, deletions, and some types of single base substitutions can be detected and their inheritance (and the inheritance of other mutations known to be closely linked) determined. Specific individuals can be uniquely identified from a modification of this technique known popularly as "DNA Fingerprinting". This third technique also begins with restriction endonuclease cleavage of genomic, cloned or PCR-amplified DNA, into fragments. The resulting fragments are separated according to size by gel electrophoresis, and certain target fragments or groups of fragments are identified by hybridization with a specific probe. In this case, the sizes of fragments identified by hybridization with the probe provide a measure of whether the target sequence complementary to the probe is part of an identical or analogous fragment from other individuals.
While each of these three techniques, and the many specific variations which have evolved from them, are extremely valuable in investigating various aspects of structure and organization of particular genes, this analysis represents only one level of genetic complexity. The ordered and timely expression of this information represents another level of complexity equally important to the definition and biology of the organism. Techniques based on complementary DNA (cDNA) subtraction or differential display can be quite useful for comparing gene expression differences between two cell types (Hedrick, et al. (1984) Nature 308:149; Liang et al. (1992) Science 257:967), but provide only a partial picture, with no direct information about abundance. The expressed sequence tag (EST) approach is a valuable tool for gene discovery (Adams et al. (1991) Science 252:1651; Adams et al. (1992) Nature 355:632; Okubo et al. (1992) Nature Genet 2:173), but like RNA blotting, ribonuclease (RNase) protection, and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis (Alwine et al. (1977) PNAS 74:5350; Zinn et al. (1983) Cell 34:865; Veres et al. (1987) Science 237:415), it evaluates only a limited number of genes at a time.
USPTO Backgrounds
USPTO Backgrounds