Solutions To Null Alleles

If a null allele is detected at a STR locus, there are several possible solutions. First, the problem PCR primer could be redesigned and moved away from the problematic site. This approach was taken early in the development of the D7S820 primers for the Promega PowerPlex® 1.1 kit (Schumm et al. 1996) and more recently with a D16S539 flanking region mutation (Nelson et al. 2002, see Chapter 5). However, this solution could result in the new primer interfering with another one in the multiplex set of primers or necessitate new PCR reaction optimization experiments. Clearly this solution is undesirable because it is time-consuming and labor intensive.

A second solution is to simply drop the STR locus from the multiplex mix rather than attempting to redesign the PCR primers to avoid the site. This approach is only desirable when early in the development cycle of a multiplex STR assay. The Forensic Science Service dropped the STR locus D19S253 from consideration in their prototype second-generation multiplex when a null allele was discovered (Urquhart et al. 1994).

A third, and more favorable, solution is to add a 'degenerate' primer that contains the known sequence polymorphism. This extra primer will then amplify alleles containing the problematic primer binding site sequence variant. This approach was taken with the AmpFlSTR® kits for the D16S539 mutation mentioned previously (Holt et al. 2002, see Chapter 5). However, if the sequence variation at the primer binding site is extremely rare, it may not be worth the effort to add an additional primer to the multiplex primer mix.

Table 6.2

Summary of discordant results observed with STR kits as reported in the literature due to various concordance studies with different PCR primer sets. These discrepancies arise due to polymorphic nucleotides or insertions/ deletions that occur in the tested DNA templates near the 3'-end of a primer binding site that disrupt proper primer annealing and result in allele dropout upon PCR amplification. STR kits reported here include: PowerPlex 1.1 (PP1.1), PowerPlex 16 (PP16), Identifier (ID), Profiler Plus (ProPlus), COfiler, and SGM Plus.

Locus

STR Kits/Assays Results

Reference

Compared

D5S818

D13S317

D16S539

D18S51

TH01

TH01

D21S11

PP1.1 us ProPlus PP16 us ProPlus ID us miniplexes SGM us SGM Plus PP16 us ProPlus ID us miniplexes ID us miniplexes

PP1.1 us PP16 us COfiler

D16S539 PP16 us COfiler D8S1179 PP16 us ProPlus D8S1179 SGM us SGM Plus

FGA SGM us SGM Plus

PP16 us ProPlus

SGM us SGM Plus

CSF1PO PP16 us COfiler

PP16 us COfiler SGM us SGM Plus PP16 us ProPlus

D19S433 SGM us SGM Plus

Loss of allele 19 with ProPlus; fine with PP1.1

Loss of alleles 15 and 17 with ProPlus; fine with PP16

Loss of alleles 12, 13, and 14 with miniplex assay; fine with ID

Loss of allele 17 with SGM Plus; fine with SGM

Loss of alleles 10 and 11 with PP16; fine with ProPlus

Loss of allele 12 with miniplex assay; fine with ID

Shift of alleles 10 and 11 due to deletion outside of miniplex assay but internal to ID

Loss of alleles with PP1.1; fine with PP16 and COfiler

Loss of allele 12 with PP16; fine with COfiler

Loss of alleles 15, 16, 17, and 18 with ProPlus; fine with PP16

Loss of allele 16 with SGM Plus; fine with SGM

Loss of allele 26 with SGM Plus; weak amp of same allele with SGM

Loss of allele 22 with ProPlus; fine with PP16

Loss of alleles 17, 18, 19, and 20 with SGM Plus (in Kuwaiti individuals); fine with SGM

Loss of allele 14 with COfiler; fine with PP16

Loss of allele 9 with COfiler; fine with PP16

Loss of allele 6 with SGM Plus; fine with SGM

Loss of allele 32.2 with PP16; fine with ProPlus

Loss of allele 11 with SGM Plus; fine with SGM

Budowle et al. (2001)

Drabek et al. (2004)

Clayton et al. (2004)

Alves et al.

Drabek et al.

Drabek et al. (2004)

Nelson et al. (2002)

Budowle et al. (2001)

Budowle et al. (2001)

Clayton et al. (2004)

Cotton et al. (2000)

Budowle and Sprecher (2001)

Clayton et al. (2004)

Budowle et al. (2001)

Budowle et al. (2001)

Clayton et al. (2004)

Budowle et al. (2001)

Clayton et al. (2004)

A fourth possible solution to correct for allele dropout that will work for some problematic primer binding sites is to re-amplify the sample with a lower annealing temperature and thereby reduce the stringency of the primer annealing. If the primer is only slightly destabilized, as detected by a peak height imbalance with a heterozygous sample (Figure 6.9, middle panel), then the peak height imbalance may be able to be corrected by lowering the annealing temperature during PCR.

No primer set is completely immune to the phenomenon of null alleles. However, when identical primer sets are used to amplify evidence samples and suspect reference samples, full concordance is expected from biological materials originating from a common source. If the DNA templates and PCR conditions are identical between two samples from the same individual, then identical DNA profiles should result regardless of how well or poorly the PCR primers amplify the DNA template.

The potential of null alleles is not a problem within a laboratory that uses the same primer set to amplify a particular STR marker. However, with the emergence of national and international DNA databases, which store only the genotype information for a sample, allele dropout could potentially result in a false negative or incorrect exclusion of two samples that come from a common source.

Heterozygous alleles are well balanced

Imbalance in allele peak heights

Was this article helpful?

0 0
Stammering Its Cause and Its Cure

Stammering Its Cause and Its Cure

This book discusses the futility of curing stammering by common means. It traces various attempts at curing stammering in the past and how wasteful these attempt were, until he discovered a simple program to cure it. The book presents the life of Benjamin Nathaniel Bogue and his struggles with the handicap. Bogue devotes a great deal of text to explain the handicap of stammering, its effects on the body and psychology of the sufferer, and its cure.

Get My Free Ebook


Post a comment