IDENTIFYING SPECIFIC BIOLOGICAL MOLECULES

Identifying DNA Sequences

Select DNA sequences30 are identified in complex mixtures (e.g., whole genomes) through the use of PCR or Southern blot.31 PCR was described previously. It entails the amplification of a target gene of interest using specially designed primers and the purified cellular machinery for DNA replication. The production of a PCR product indicates the presence of the sequence of interest within the sample. Thus, the results are a “yes” or “no” answer. Since primers are short (~20 bp), any mismatch between a primer and its target template sequence compromises the reaction. Therefore PCR is not an ideal method for the detection of sequences that are similar, but not identical. For example, suppose you were interested in learning how often a particular antibiotic resistance gene is found within the genomes of moderately related bacterial strains or species.32 PCR would not be a good method to use because modest differences (e.g., single nucleotide polymorphisms, SNPs) are likely to exist among bacteria, as a result of evolutionary distance. Thus, primers might fail to bind to some versions of the gene, PCR products would not be made, and false negative results would be obtained.

A technique called Southern blot is a better choice for identifying similar but not identical DNA sequences. Like PCR, Southern blot utilizes the binding of complementary bait sequences (in PCR, referred to as primers; in Southern blot, referred to as probes) to detect target sequences of interest (Southern 1975). Unlike PCR, the complementary probe sequences of Southern blots are quite large (~200+ bp), which makes the binding of bait to target more tolerant of mismatches. Thus, Southern blots are a powerful tool for cross-strain, -species, or even -genera comparisons.

In Southern blot, a complex DNA sample (e.g., a whole genome, chromosome, or plasmid) is isolated and fragmented by one or more restriction enzymes. The fragments are separated by DNA agarose gel electrophoresis and transferred from the gel onto a membrane such as nitrocellulose. DNA fragments are immobilized onto the surface of the membrane by UV-mediated or other mechanisms of crosslinking. Next, a labeled single-stranded DNA probe (usually made by denaturing a PCR product) is washed over the surface of the membrane. The probe binds (i.e., sticks, hybridizes, anneals) to complementary DNA within the sample. Unbound probe is removed by washing. Since the probe is labeled with radioactive P32 or another detectable chemical moiety (e.g., biotin), the presence of bound probe can be detected using methods such as autoradiography. Generation of a “band” within a sample lane is indicative of the presence of the target sequence.

 
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