Polymerase chain reaction - An Introduction to Forensic Genetics - page 55

polymerase (Applied Biosystems). The enzyme is inactive when it is first added to the
PCR; it only becomes active after incubation at 95 ◦ C for approximately 10 minutes
[23]. This 'hot start' enzyme allows the PCR to start at an elevated temperature and
minimizes the non-specific binding that can occur at lower temperatures [24].
Primers
The primers used in PCR define the region of the genome that will be analysed.
Primers are short synthetic pieces of DNA that anneal to the template molecule
either side of the target region. The primer sequences are therefore limited to some
degree by the DNA sequence that flanks the target sequence. Figure 5.1 illustrates
the sequence and positioning of two primers that are used to amplify the D16S539
locus in one of the commercially available kits for analysing STR loci (Promega
Corporation).
When designing primers for forensic analysis it is important that they will bind to
conserved regions of DNA and therefore effectively amplify human DNA from all
populations [25], while at the same time not binding to the DNA of other species.
When designing a multiplex PCR, the allelic size ranges are also important consid-
erations for the position of the primer binding sites.
There are a number of basic guidelines for primer design. Primers are normally
between 18 and 30 nucleotides long, and have a balanced number of G/C and A/T
nucleotides. A primer should not be self-complementary or be complementary to any
of the other primers that are in the reaction. Self-complementary regions will result in
the primer pairing with itself to form a loop, whereas primers that are complementary
will bind to each other to form primer dimers.
The temperature at which primers anneal to the template DNA depends upon
their length and sequence; most primers are designed to anneal between 50 ◦ Cand
65 ◦ C. A basic rule of thumb can be used when designing primers to estimate the
melting temperature (the temperature at which half of a particular DNA duplex
01 ggagctg ggg ggtctaagag cttgtaaaaa g tgtacaagt gccagatgct cgttgtgcac
61 aaatctaaat gcagaaaagc actgaaagaa gaatcccgaa aaccacagtt cccattttta
121 tatgggagca aacaaagcag atcccaagct cttcctcttc cctagatcaa tacagacaga
181 cagacaggtg GATAGATAGA TAGATAGATA GATAGATAGA TAGATAGATA GATA tcattg
241 aaagacaaaa cagagatgga tgatagatac atgcttacag atgcacacac aaac gctaaa
Forward Primer: 5 ′ - GGG GGT CTA AGA GCT TGT AAA AAG - 3 ′
Reverse Primer: 5 ′ - GTT TGT GTG TGC ATC TGT AAG CAT - 3 ′
Figure 5.1 The forward and reverse primers that are used to amplify the short tandem repeat (STR)
locus D16S539 in the Promega Powerplex 1.2 Kit are shown; the position that they bind to within
the sequence is indicated by the arrows. The target sequence is shown in bold: the position of the
primers around either side of the target determines the length of the PCR product. The example
contains 11 repeats of the core GATA sequence (see Chapter 6 for details of STRs)