Advanced Techniques in Diagnostic Microbiology

11. PCR and Its Variations 179
presence of repetitive sequences in a wide range of bacterial species and demonstrated
their use to directly fingerprint bacterial genomes (Fig. 11.5). Specific
repetitive sequences include the 124–127 base-pair ERIC sequence, the 154 basepair
BOX sequence, and the 35–40 base-pair repetitive extragenic palindromic sequence.
These sequences are located intergenically throughout the chromosome.
Some repetitive sequences translocate to new locations in the genome and are called
transposons or insertion sequences. Some ISs are species-specific, whereas others
have no species restriction. VNTR’s are repeated sequences of non-coding DNA.
Whether ERIC, IS, VNTR, or other repetitive element or sequence, the basis of
the strain typing is the same. The ability of repetitive element–based PCR methods
to distinguish unrelated strains or species is based on the random distribution of
elements within the genome and the time required for these to become established.
That is, all bacteria associated with a common source outbreak are highly unlikely
to have any differences in the number or location of repetitive elements, whereas
bacteria that are geographically, temporally, and epidemiologically unrelated are
more likely to have experienced mutational events. Repetitive element–based PCR
assays are designed so that primers anneal to the specific sequence in an outward
orientation, so that DNA between the repeated elements is amplified. Variability
between unrelated organisms is due to the random number and location of the
elements on the genome.
Appendix 1
Preparation of PCR Reaction
The PCR master mix contains all of the components necessary to make new strands
of DNA in the PCR process. The master mix reagents include:
Final Conc. Component Purpose
1X Buffer Maintains proper pH of the PCR reaction.
200 μM Deoxynucleotides Provide both the energy and nucleosides for the synthesis of
DNA. It is important to add equal amounts of each
nucleotide (dATP, dTTP, dCTP, dGTP) to the master mix to
prevent mismatches of bases.
0.2–1.0 μM Primers Short pieces of DNA (20–30 bases) that bind to the DNA
template allowing Taq DNA polymerase enzyme to initiate
incorporation of the deoxynucleotides. Both specific and
universal (arbitrary) primers can be used.
1.5–2.5 μU Taq polymerase A heat-stable enzyme that adds the deoxynucleotides to the
DNA template.