The amount of stutter product formation may be reduced when using STR markers with longer repeat units, STR alleles with imperfect repeat units, and DNA polymerases with faster processivity. Several pentanucleotide repeat loci have been developed in an effort to produce STR markers that exhibit low amounts of stutter products to aid in mixture interpretation (Bacher and Schumm 1998). The first seven loci have been labeled Penta A through Penta G. Penta E has been incorporated in the GenePrint® PowerPlex™ 2.1 system and reportedly exhibits an average stutter percentage of less than 1% (Bacher et al. 1999). Both Penta D and Penta E are part of the PowerPlex® 16 kit (Krenke et al. 2002).
Alleles for a STR locus that contain variations on the common repeat motif exhibit a smaller amount of stutter product formation. For example, the common repeat motif for the STR marker TH01 is AATG. However, with allele 9.3, there is an ATG nucleotide sequence present in the middle of the repeat region (Puers et al. 1993). When the core repeat sequence has been interrupted, stutter product formation is reduced compared to alleles that are similar in length but possess uninterrupted core repeat sequences. This fact has been demonstrated with sequencing results from several VWA alleles (Walsh et al. 1996).
The amount of stutter may be related to the DNA polymerase processivity, or how rapidly it copies the template strand. Stutter products have been shown to increase relative to their corresponding alleles with a slower polymerase (Walsh et al. 1996). If thermal stable DNA polymerases become available in the future that are faster than the current 50-60 base processivity of Taq DNA polymerase, then it may be possible to reduce the stutter product formation. A faster polymerase would be able to copy the two DNA strands before they could come apart and re-anneal out of register during primer extension (see Figure 6.2).
A summary of stutter product formation is listed below:
■ Primarily one repeat unit smaller than corresponding main allele peak;
■ Typically less than 15% of corresponding allele peak height;
■ Quantity of stutter depends on locus as well as PCR conditions and polymerase used;
■ Propensity of stutter decreases with longer repeat units (pentanucleotide repeats<tetra-<tri-<dinucleotides);
■ Quantity of stutter is greater for large alleles within a locus;
■ Quantity of stutter is less if sequence of repeats is imperfect.
DNA polymerases, particularly the Taq polymerase used in PCR, often add an extra nucleotide to the 3'-end of a PCR product as they are copying the template strand (Clark 1988, Magnuson et al. 1996). This non-template addition is most often adenosine and is therefore sometimes referred to as 'adenylation' or the '+A' form of the amplicon. Non-template addition results in a PCR product that is one base pair longer than the actual target sequence.
Addition of the 3' A nucleotide can be favored by adding a final incubation step at 60°C or 72°C after the temperature cycling steps in PCR (Clark 1988, Kimpton et al. 1993). However, the degree of adenylation is dependent on the sequence of the template strand, which in the case of PCR results from the 5'-end of the reverse primer (Figure 6.4). If the forward primer is labeled with
(a) with illustrated measurement result (b). DNA polymerases add an extra nucleotide beyond the 3-end of the target sequence extension product. The amount of non-template addition is dependent on the sequence of the 5'-end of the opposing primer. In the case of dye labeled PCR products where the fluorescent dye is on the forward primer, the reverse primer sequence is the critical one.
Ways to convert STR allele peaks to either —A or +A forms.
a fluorescent dye to amplify the STR allele, then only the top strand is detected by the fluorescent measurement. Since the sequence at the 3'-end of the top (labeled) strand serves as a template for polymerase extension, the terminal nucleotide of the labeled strand is determined by the 5'-end of the reverse primer used in generating the complementary unlabeled strand (Magnuson et al. 1996). One study found that if the 5'-terminus of the primer is a guanosine, then a complete addition is favored by the polymerase (Brownstein et al. 1996). Thus, every locus will have slightly different adenylation properties because the primer sequences differ.
Now why is all of this important? From a measurement standpoint, it is better to have all of the molecules as similar as possible for a particular allele. Partial adenylation, where some of the PCR products do not have the extra adenine (i.e., —A peaks) and some do (i.e., +A peaks), can contribute to peak broadness if the separation system's resolution is poor. Sharper peaks improve the likelihood that a system's genotyping software can make accurate calls. In addition, variation in the adenylation status of an allele across multiple samples can have an impact on accurate sizing and genotyping potential microvariants. For example, a non-adenylated TH01 10 allele would be the same size as a fully adenylated TH01 9.3 allele because they contain an identical number of base pairs. Therefore, it is beneficial if all PCR products for a particular amplification are either +A or —A rather than a mixture of +/—A products. Table 6.1 lists some of the methods that have been used to convert PCR products into either the —A or +A form.
Final extension at 60°C or 72°C for 30-45 minutes
Conversion to fully adenylated products (+A form)
Promotes full adenylation of all products
Kimpton et al. 1993, Applied Biosystems 1999
Addition of sequence GTTTCTT on the Promotes nearly 100% adenylation of the 3' 5'-end of reverse primers ('PIG-tailing') forward strand
Brownstein et al. 1996
Conversion to blunt-ended products (-A form)
Restriction enzyme site built into reverse primer
Makes blunt end fragments following restriction Edwards et al. 1991 enzyme digestion
Enzymatic removal of one base overhang
Exonuclease activity of Pfu or T4 DNA polymerase removes +A
Ginot et al. 1996
Use of modified polymerase without Polymerase does not add 3' A nucleotide terminal transferase activity
During PCR amplification most STR protocols include a final extension step to give the DNA polymerase extra time to completely adenylate all double-stranded PCR products. For example, the standard AmpF/STR® kit amplification parameters include a final extension at 60°C for 45 minutes at the end of thermal cycling (Applied Biosystems 1999). In order to make correct genotype calls, it is important that the allelic ladder and the sample have the same adeny-lation status for a particular STR locus. For all commercially available STR kits, this means that the STR alleles are all in the +A form.
Amplifying higher quantities of DNA than the optimal amount suggested by the manufacturer's protocols can result in incomplete 3' A nucleotide addition and therefore split peaks. The addition of 10 ng of template DNA to a PCR reaction with AmpFKTR Profiler Plus results in split peaks compared to using only 2ng of the same template DNA (Figure 6.5). Thus, quantifying the amount of DNA prior to PCR and adhering to the manufacturer's protocols will produce improved STR typing results with using commercial STR kits.
MICROVARIANTS AND 'OFF-LADDER' ALLELES
Rare alleles are encountered in the human population that may differ from common allele variants at tested DNA markers by one or more base pairs. Sequence variation between STR alleles can take the form of insertions, deletions, or nucleotide changes. Alleles containing some form of sequence variation compared to more commonly observed alleles are often referred to as microvariants because they are only slightly different from full repeat alleles. Because microvariant alleles often do not size the same as consensus alleles present in the reference allelic ladder, they can be referred to as 'off-ladder' alleles.
120 130 140 150 160 170 180 190 200 210 220 230 -1-1-1-1-1-1-1-1-1-1— i
60004000200006000 4000 2000
10 ng template (overloaded)
D3S1358 VWA FG
2 ng template
JU1 it (suM~ j
Incomplete non-template addition with high levels of DNA template. In the top panel, partial adeny-lation (both —A and +A forms of each allele) is seen because the poly-merase is overwhelmed due to an abundance of DNA template. Note also that the peaks in the top panel are off-scale and flat-topped in the case of the smaller FGA allele. When the suggested level of DNA template is used, all alleles are fully adeny-lated (bottom panel).
One example of a common microvariant is allele 9.3 at the STR locus TH01. The repeat region of TH01 allele 9.3 contains nine full repeats (AATG) and a partial repeat of three bases (ATG). The 9.3 allele differs from the 10 allele by a single base deletion of adenine in the seventh repeat (Puers et al. 1993).
Microvariants exist for most STR loci and are being identified in greater numbers as more samples are being examined around the world. In a recent study, 42 apparent microvariants were seen in over 10 000 samples examined at the CSF1PO, TPOX, and TH01 loci (Crouse et al. 1999). Microvariants are most commonly found in more polymorphic STR loci, such as FGA, D21S11, and D18S51, that possess the largest and most complex repeat structures compared to simple repeat loci, such as TPOX and CSF1PO (see Appendix I).
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