Assessment of STR profiles

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DNA profiles generated from casework samples require some experience to interpret. Guidelines have evolved to assist with the interpretation of STR profiles, ensuring that the results are robust and consistent; this is especially important when dealing with samples that contain very small amounts of DNA, degraded DNA or mixtures of profiles that come from two or more individuals - all situations that complicate interpretation. This chapter explores a number of artefacts that can occur in DNA profile. Some casework scenarios that can lead to complex profiles are also considered.

Stutter peaks

During the amplification of an STR allele it is normal to generate a stutter peak, that is one repeat unit smaller or larger that the true allele; smaller alleles are formed in the majority of cases [1]. Stutter peaks are formed by strand slippage during the extension of the nascent DNA strand during PCR amplification (Figure 7.1) [2, 3].

Even in good quality profiles there will be some stutter peaks; these are recognizable and do not interfere with the interpretation of the profile. Threshold limits are normally used to aid in the identification and interpretation of stutter peaks, so, for example, while the degree of stutter varies between loci, they are typically less than 15 % of the main peak [4,5]- understanding stutter peaks is especially important when interpreting mixtures.

Different STR loci have varying tendencies to stutter. This is dependent on the structure of the core repeats: shorter di- and trinucleotide repeats are more prone to stutter than are tetra- and pentanucleotide repeats and this is one of the reasons that all the autosomal STRs that have been adopted by the forensic community have tetra and pentanucleotide core repeats (Figure 7.2). STRs with simple core repeats tend to have higher stutter rates than compound and complex repeats.

The Taq polymerase that is used to drive the polymerase chain reaction adds nucleotides to the newly synthesized DNA molecule in a template-dependent manner. However, it

Template strand

AGAAAGAAAGAAAGAA

Extension

Nascent strand

Figure 7.1 During PCR, slippage between the template and the nascent DNA strands leads to the copied strand containing one repeat less than the template strand also has an activity, called terminal transferase, whereby it adds a nucleotide to the end of the amplified molecule which is non-template-dependent [6]. Approximately 85 % of the time an adenine residue is added (Figure 7.3).

It is important that the vast majority of PCR products have the non-template nucleotide added, otherwise a split peak is observed in the DNA profile (Figure 7.4). Split peaks are usually caused either by the sub-optimal activity of the Taq polymerase or by too much template DNA in the PCR.

In order to minimize the formation of split peaks in a profile, at the end of the cycling stage of the PCR, the reaction is incubated at 65-72 °C for between 45-60 minutes, allowing the Taq polymerase to complete the non-template addition of all the PCR products.

The interpretation of profiles with split peaks is possible because the peak with the nucleotide added is taken as being the correct peak. Problems can occur when alleles are present that differ by only one base pair; the THO1 9.3 allele for example could be confused with the THO1 allele 10. In most cases a profile with a high degree

Polymerase Stutter
2 bp

4 bp

4 bp

Figure 7.2 Stutter peaks are formed during slippage of the Taq polymerase during replication of the template strand. The slippage results in amplification products one repeat unit shorter than the template. The stutter peaks are normally less than 15% of the true amplification product. Panel

(a) shows a dinucleotide repeat, which is prone to high levels of slippage, the stutter peaks are indicated by the arrow and their size relative to the main peak is shown (based on peak area). Panel

(b) is a tetranucleotide repeat, which displays lower levels of stutter

2 bp

2 bp

4 bp

4 bp

Figure 7.2 Stutter peaks are formed during slippage of the Taq polymerase during replication of the template strand. The slippage results in amplification products one repeat unit shorter than the template. The stutter peaks are normally less than 15% of the true amplification product. Panel

(a) shows a dinucleotide repeat, which is prone to high levels of slippage, the stutter peaks are indicated by the arrow and their size relative to the main peak is shown (based on peak area). Panel

(b) is a tetranucleotide repeat, which displays lower levels of stutter

PULL-UP

Template

PCR product

Figure 7.3 The Taq polymerase adds a nucleotide to the 3' end of the newly synthesized strand. The non-template addition is usually an adenine and results in a PCR product that is one base pair longer than the template (N + 1). The arrowed lines represent the forward and reverse primers of split peaks would have to be re-analysed to minimize the possibility of incorrect interpretation.

Pull-up

In Chapter 6 the matrix file was introduced - this file contains information about the levels of spectral overlap that exist with the dyes that have been used to label the PCR products. This information is used by the Genescan® and GeneMapper™ ID software to produce peaks that are made up of one colour. If the matrix file is not of good quality then this correction is not perfect and the peaks in the resulting profile are composed of more than one colour; this phenomenon is called pull-up. Pull-ups are easy to recognize as a smaller sized product will appear at exactly the same size as the real STR allele. Pull-up can also occur when there has been over amplification, even if the matrix file is of good quality (Figure 7.5a).

4000 3500 3000 2500 2000 1500 1000 500 0

4000 3500 3000 2500 2000 1500 1000 500 0

Figure 7.4 Split peaks are seen in profiles when the non-template addition does not occur with all of the PCR products. The three examples show decreasing amounts of non-template addition with panel (a) showing an example where the vast majority of PCR product has the non-template addition through to panel (c), where only 50% of the PCR product has the non-template addition

(a) 25 ng template

^-Split peaks

2,000 rfu

(c) 1 ng template

1,000 rfu

0.25 ng template

Allele drop-out

Allele drop-out

50 rfu

0.25 ng template

Allele drop-out

Allele drop-out

50 rfu

Figure 7.5 If the reaction is overloaded with DNA, (a) the peaks are still present but artefacts such as pull-ups and split peaks are more pronounced. When the template is within the optimal range (b and c) the peaks are well balanced and easy to interpret. When the PCR does not have enough template to amplify (d), then locus and allelic drop-out can occur (see plate section for full-colour version of this figure)

Figure 7.5 If the reaction is overloaded with DNA, (a) the peaks are still present but artefacts such as pull-ups and split peaks are more pronounced. When the template is within the optimal range (b and c) the peaks are well balanced and easy to interpret. When the PCR does not have enough template to amplify (d), then locus and allelic drop-out can occur (see plate section for full-colour version of this figure)

Template DNA

Commercial STR kits have been optimized to amplify small amounts of template DNA, commonly between 0.5 and 2.5 ng, which represents approximately 166 and 833 copies of the haploid human genome. It is not always possible to add the optimum amount of DNA to a PCR when the sample size is limited.

Overloaded profiles

Overloading the PCR can also lead to a profile that is difficult to interpret. If the CCD camera is saturated then the peak height/area is no longer a good indicator of the amount of product and this can lead to problems in assessing peak balance and can make the interpretation of mixtures difficult. Overloaded profiles also tend to have a noisy baseline, increased levels of stuttering, split peaks and pull-ups (Figure 7.6).

Low copy number DNA

At many crime scenes it may be possible to infer surfaces with which the perpetrator has had physical contact, for example the handle of a gun, a knife, a ligature, a door handle or a steering wheel. These areas can be swabbed to collect any epithelial cells that have been shed during the contact [7-10]. The amounts of DNA extracted can be extremely low but in some circumstances it is possible to get a full DNA profile from less than 100 pg of template DNA: the normal range of template DNA is between 500 and 2500 pg (2.5 ng). To analyse such small quantities of DNA the number of amplification cycles is increased to 34. The standard number of cycles in the amplification using commercial kits is between 28 - 32 cycles. Empirical studies have shown that above 34 cycles the

LOW COPY NUMBER DNA

LOW COPY NUMBER DNA

Forensic Low Copy Number

Figure 7.6 A heavily overloaded profile. AH the peaks shown have a flat top indicating that they are off-scale, the baseline is very noisy, several split peaks are evident and the peaks are very broad, which can lead to sizing problems. There are also some pronounced stutter peaks (see plate section for full-colour version of this figure)

Figure 7.6 A heavily overloaded profile. AH the peaks shown have a flat top indicating that they are off-scale, the baseline is very noisy, several split peaks are evident and the peaks are very broad, which can lead to sizing problems. There are also some pronounced stutter peaks (see plate section for full-colour version of this figure)

amount of artefacts that are detected outweigh the benefit of higher levels of artefact [11]. Extreme care has to be taken when interpreting the LCN profiles [11,12]. A number of features can be seen when amplifying low amounts of template DNA. These are: allele drop-out, and drop-in; severe peak imbalance; locus drop-out (Figure 7.5d); and increased stutter [12-14]. Allele drop-out occurs when through chance events one allele in a heterozygous locus is preferentially amplified; this can give the false impression that the profile at a particular locus is homozygous. To minimize the possibility of this occurring the PCR must be repeated at least two times and only alleles that appear consistently can be called (Figure 7.7).

This phenomenon also leads to a peak imbalance that is much higher than when using higher amounts of template DNA. Allele drop-in is also a common phenomenon when amplifying low amounts of template DNA. The drop-in alleles are spurious amplification products and are not amplified in the duplicate or triplicate reactions but can still confuse the interpretation of the profile. Locus drop-out, particularly of the larger STR loci, can also occur; this reduces the amount of information from the profile but does not confuse the interpretation. At present, there is no clear consensus in the scientific community about the use of LCN PCR [15].

D21S11 D21S11 D21S11

D21S11 D21S11 D21S11

Figure 7.7 Three separate PCR analyses of a DNA extract can lead to different results. When dealing with very low template numbers, allelic dropout is relatively common and the true genotype can only be ascertained through multiple amplifications - and even then the results can be contentious

Figure 7.7 Three separate PCR analyses of a DNA extract can lead to different results. When dealing with very low template numbers, allelic dropout is relatively common and the true genotype can only be ascertained through multiple amplifications - and even then the results can be contentious

IfEQ.

Amelogenin D8S1179

D21S11

D18S51

Figure 7.8 The profile shows the green locifrom the AmpF/STR® SGM Plus® kit. The peak area of the smallest peak at each locus is shown as a percentage of the larger peak. The size of the peaks is proportional to the amount of PCR product - this can be gauged by measuring the peak height or, more usually, the peak area

Peak balance

STR loci that are used in forensic analysis are commonly heterozygous, producing two peaks in the profile. In a perfect profile the two peaks that are produced are balanced 1:1 in terms of peak height and area but in reality this is very rare and one peak will be larger than the other (Figure 7.8). The variations in peak height can be due to chance events, where one allele is more efficiently amplified than another. In good quality DNA extracts, the smaller peak is, on average, approximately 90 % the size of the larger peak [4].

Laboratories will use different values that are based on their own validation studies but commonly require the smaller peak of a locus to be within 60 % of the larger peak [4]. Peak imbalance can be more extreme when profiling degraded DNA and when amplifying low amounts of template DNA. On rare occasions the mutation of a primer binding site will reduce the efficiency of the PCR for one allele, which can result in high levels of peak imbalance and even allele drop-out. The frequency of these mutations is low, ranging between frequencies of 0.01 and 0.001 per locus [16].

Mixtures

Many biological samples that are recovered from a scene of crime will contain a mixture of cellular material from more than one person. Clothing will often contain cellular material from the wearer and may also contain material from an assailant after an assault; the handle of a door or a steering wheel may have been handled by several people - there are many circumstances when mixtures of material can be collected. A mixture in a DNA profile can be recognized by the presence of more than two alleles at any locus within the profile, normally there will be several loci that have three or four alleles present and a loss of peak balance.

Having determined that the profile is mixed, the first task is to assess how many contributors are represented in the profile. Two person mixtures are most commonly seen in forensic casework; with a two person mixture a maximum of four alleles will be present at any locus whereas three person mixtures will contain up to six alleles

DEGRADED DNA

DEGRADED DNA

Figure 7.9 A mixture of two individuals will lead to up to four peaks at each locus. The area between the dotted lines represents the zone where the minor component of the mixture can be interpreted. The lower dotted line represents 15 % and 60 % of the major peaks - below 15 % is a zone where stutter peaks from the major alleles can occur and peaks, below 60 % cannot be easily explained by peak imbalance. At this locus the major component can be interpreted as 13-15 while the minor component's genotype is 16 - 20

Figure 7.9 A mixture of two individuals will lead to up to four peaks at each locus. The area between the dotted lines represents the zone where the minor component of the mixture can be interpreted. The lower dotted line represents 15 % and 60 % of the major peaks - below 15 % is a zone where stutter peaks from the major alleles can occur and peaks, below 60 % cannot be easily explained by peak imbalance. At this locus the major component can be interpreted as 13-15 while the minor component's genotype is 16 - 20

at a locus. When four alleles are present at a given locus and there is a major and minor component, the interpretation is relatively simple (Figure 7.9). The ratio of peak areas within a locus generally corresponds with the ratio of template molecules [17, 18], peak areas that consider the morphology of the peak as well as the height [16], are commonly used as a guide to interpret mixed profiles [19]. Even in a two-person mixture when there are shared alleles between the major and minor profiles, the interpretation becomes more difficult - especially in mixtures where the minor profile is less than one-third of the level of the major profile [16].

In mixtures where the major component is in large excess, it is often possible to deduce the major profile; however, in such cases it is difficult to get much information from the minor component where the interpretation is complicated by artefacts in the profile such as stutter peaks, and also by the major profile masking the minor profile [20]. Software has been developed that helps with the interpretation of complex mixtures [21].

Degraded DNA

Many samples that are collected from a crime scene may have been exposed to the environment for hours, days, or even longer if the crime scene has gone undetected. When DNA analysis is being used to identify human remains, the remains may be several years old before they are analysed or may have been exposed to severe environmental insult such as high temperatures. In all these circumstances the DNA in the

Bone Str Profile

Figure 7.10 The profile was generated using the AmpF/STR® Profiler Plus® kit from Applied Biosystems. The DNA was extracted from a bone recovered from a Scottish loch after approximately 30 years. The profile is typical of a degraded profile with a gradual reduction in the amount of product as the amplicons increase in size (see plate section for full-colour version of this figure)

Figure 7.10 The profile was generated using the AmpF/STR® Profiler Plus® kit from Applied Biosystems. The DNA was extracted from a bone recovered from a Scottish loch after approximately 30 years. The profile is typical of a degraded profile with a gradual reduction in the amount of product as the amplicons increase in size (see plate section for full-colour version of this figure)

cellular material will not be in pristine condition and will have degraded. This leads to a characteristic DNA profile with over amplification of the smaller loci and the successful amplification declines with the size of the alleles. Figures 7.10 and 7.11 show two examples of degraded DNA sample; the first one is from a bone sample that had been in water for 30 years. The small loci have over amplified whereas the larger loci are barely detectable [22] - the decrease in amplification is gradual as the length of the alleles increases.

In the second example an example of locus drop out can be seen, the first two blue loci, D3S1358 and vWA have amplified successfully but there is no FGA allele. This

2400

270C 2 IOC.

1 50C

BOC.

60C 3DC

D3S1358

Figure 7.11 The profile was generated using the AmpF/STR Blue™ kit from Applied Biosystems. The DNA was extracted from muscle tissue recovered from a plane crash. The muscle had been subjected to high temperatures and the DNA was highly degraded - no amplification products were detected from the FGA locus. The size standard is also shown in by non-shaded peaks (see plate section for full-colour version of this figure)

REFERENCES

profile is from human muscle tissue that had been exposed to high temperatures and has degraded to the extent that there is very little or no DNA that is 200 bp or longer [23]. The interpretation of degraded profiles can be difficult and particular attention has to be taken when homozygous loci are detected - are they really homozygous and not heterozygous with one of the alleles having dropped out? When the levels are very low, if there is enough material the PCR is carried out in duplicate, as with LCN PCR, to minimize the possibility of generating an incorrect profile.

To assist with the analysis of degraded DNA, a series of multiplexes have been developed with the primers positioned close to the core repeats of the STRs, thereby minimizing the lengths of the amplicons [24-28].

References

1. Shinde, D., et al. (2003) Taq DNA polymerase slippage mutation rates measured by PCR and quasi-likelihood analysis: (CA/GT)(n) and (A/T)(n) microsatellites. Nucleic Acids Research 31, 974-980.

2. Hauge, X.Y., and Litt, M. (1993) A study of the origin of shadow bands seen when typing dinucleotide repeat polymorphisms by the Pcr. Human Molecular Genetics 2, 411-415.

3. Schlotterer, C., and Tautz, D. (1992) Slippage synthesis of simple sequence DNA. Nucleic Acids Research 20, 211-215.

4. Gill, P., et al. (1997) Development of guidelines to designate alleles using an STR multiplex system. Forensic Science International 89, 185-197.

5. Corporation, T.P.-E. (1999) AmpFISTR SGM Plus™ PCR Amplification Kit - User's Manual.

6. Clark, J.M. (1988) Novel non-templated nucleotide addition-reactions catalyzed by procaryotic and eukaryotic DNA-polymerases. Nucleic Acids Research 16, 9677-9686.

7. Bohnert, M., et al. (2001) Transfer of biological traces in cases of hanging and ligature strangulation. Forensic Science International 116, 107-115.

8. Esslinger, K.L., et al. (2004) Using STR analysis to detect human DNA from exploded pipe bomb devices. Journal of Forensic Sciences 49, 481-484.

9. van Oorschot, R.A.H., and Jones, M.K. (1997) DNA fingerprints from fingerprints. Nature 387, 767-767.

10. Wiegand, P., and Kleiber, M. (1997) DNA typing of epithelial cells after strangulation. International Journal of Legal Medicine 110, 181-183.

11. Gill, P. (2001) Application of low copy number DNA profiling. Croatian Medical Journal 42, 229-232.

12. Gill, P., et al. (2000) An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA. Forensic Science International 112, 17-40.

13. Kloosterman, A.D., and Kersbergen, P. (2003) Efficacy and limits of genotyping low copy number DNA samples by multiplex PCR of STR loci. International Congress Series 1239, 795-798.

14. Whitaker, J.P., et al. (2001) A comparison of the characteristics of profiles produced with the AMPFlSTR® SGM PlusTM multiplex system for both standard and low copy number (LCN) STR DNA analysis. Forensic Science International 123, 215-223.

15. Gill, P., et al. (2006) DNA commission of the International Society of Forensic Genetics: recommendations on the interpretation of mixtures. Forensic Science International 160, 90-101.

16. Clayton, T.M., et al. (1998) Analysis and interpretation of mixed forensic stains using DNA STR profiling. Forensic Science International 91, 55-70.

17. Lygo, J.E., et al. (1994) The validation of short tandem repeat (Str) loci for use in forensic casework. International Journal ofLegal Medicine 107, 77-89.

18. Sparkes, R., et al. (1996) The validation of a 7-locus multiplex STR test for use in forensic casework.1. Mixtures, ageing, degradation and species studies. International Journal of Legal Medicine 109, 186-194.

19. Evett, I.W., et al. (1998) Taking account of peak areas when interpreting mixed DNA profiles.

Journal of Forensic Sciences 43, 62-69.

20. Gill, P., et al. (1998) Interpreting simple STR mixtures using allele peak areas. Forensic Science International 91, 41-53.

21. Bill, M., et al. (2005) PENDULUM-a guideline-based approach to the interpretation of STR mixtures. Forensic Science International 148, 181-189.

22. Goodwin, W., et al. (2003) The identification of a US serviceman recovered from the Holy Loch, Scotland. Science and Justice 43, 45-47.

23. Goodwin, W., et al. (1999) The use of mitochondrial DNA and short tandem repeat typing in the identification of air crash victims. Electrophoresis 20, 1707-1711.

24. Dixon, L.A., et al. (2006) Analysis of artificially degraded DNA using STRs and SNPs - results of a collaborative European (EDNAP) exercise. Forensic Science International 164, 33-44.

25. Gill, P., et al. (2006) The evolution of DNA databases - recommendations for new European STR loci. Forensic Science International 156, 242-244.

26. Coble, M.D., and Butler, J.M. (2005) Characterization of new MiniSTR loci to aid analysis of degraded DNA. Journal ofForensic Sciences 50, 43-53.

27. Drabek, J., et al. (2004) Concordance study between Miniplex assays and a commercial STR typing kit. Journal ofForensic Sciences 49, 859-860.

28. Butler, J.M., et al. (2003) The development of reduced size STR amplicons as tools for analysis of degraded DNA. Journal ofForensic Sciences 48, 1054-1064.

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