Non-human DNA contamination in Forensics

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sensitivity, specificity as well as for addressing major issues of forensic samples such as non-human DNA contamination and PCR inhibitors. In this context, the PEC- based enrichment strategy described in this study has demonstrated the efficiency of the technique to address the deleterious effects of exogenous components on STR-PCR. Additionally, the biotin oligos which were designed to bind as close as possible to the repeat regions of target loci enable the enrichment method to capture the target templates even in case of considerable degradation, much akin to the mini-STR strategy employed for heavily degraded DNA samples.

3.2.3 Mixture of inhibitors:

The sequence- specific hybrid capture methodology was further evaluated on simulated samples representing extremely compromised forensic specimens involving the co-occurrence of various PCR inhibitors and non-human DNA.

Under natural conditions, it is not unusual to find forensic specimens to be contaminated with multiple PCR inhibitors and non-human DNAs concurrently. Highly compromised samples such as buried bones could be associated with multiple inhibitors such as calcium, humic acid, tannic acid and also the exogenous DNA from microorganisms (hematin which is widely found in blood samples was not tested in these experiments). Samples prepared to emulate such real time effects of contamination as described earlier, when subjected to PEC-based enrichment demonstrated the successful generation of DNA profiles in the presence of up to 0.06 µg/µl of humic acid, 0.225 µg/µl of tannic acid and 0.6 mM of calcium. These values represent ~15% of the maximum individual tolerance limits that the PEC- based enrichment strategy was able to generate profiles against each of these inhibitors. Even though the simulated samples constituting all these 3 inhibitors beyond the above mentioned concentrations resulted in allele/locus drop outs, the amplification achieved with the aid of of selective enrichment is several folds higher than the conventional procedures.

When non-human DNA was doped in a ratio of 1:10000 (20 μg of bacterial DNA with 2 ng of sheared human DNA), in presence of the above three inhibitors, complete STR profiles were observed and surprisingly with greater peak heights (Figure 3), which may be explained on the assumption of the negation of the effect of PCR inhibitors by non-human DNA due to dilution effect. The effective concentration and therefore the influence of the PCR inhibitors on the Taq polymerase is diluted out in the presence of large number of non-human DNA molecules in the reaction assay. Encouraged by the positive outcomes from the above experiments, the STR capture technique was applied to some of the compromised forensic case work samples from which the DNA profiles could not be generated previously.

3.3 Application of STR-enrichment technique on challenging forensic samples:

Out of seven forensic samples which were examined in this experiment, three samples failed to amplify whereas remaining 4 samples generated partial DNA profiles with low peak heights when amplified either by AmpFlSTR® Identifiler® PCR amplification kit or Powerplex® 16 HS system. However, when these samples were subjected to modified PEC based STR capture, the undermined template molecules got enriched, generating a complete profile from one sample while increasing the number of amplified loci with greater peak heights from the other five samples (Table 3). Further observation of the amplification pattern indicated that the samples previously showing locus dropouts at loci with larger amplicon lengths, displayed significant improvement in their amplification upon enrichment, suggesting the ability of the technique to not only enrich the STRs of interest but also to mitigate the effect of PCR inhibitors which may preferentially affect the larger loci (26). Although the peak heights for the larger loci were lower as compared to the smaller loci, it could be attributed to the degradation of the DNA often confronted with the challenging forensic samples, a process commonly referred as ‘DNA decay’. Observation of the quantification results indicated that the samples which generated either complete or partial profiles after enrichment showed the presence of DNA either on agarose gel or by qPCR assay while samples, with no detectable DNA by any of the quantification assays failed to generate STR profiles even after enrichment. Although, it might be highly desirable to correlate the qPCR and agarose gel based quantification results to predict the probability of obtaining successful DNA profiles from forensic samples, it should be noted that the results of quantification and genotyping are affected by multiple factors and hence, accurate prediction is not always possible.

Based on this study, it can be stated that hybrid-capture method makes it possible to retrieve pristine DNA molecules from contaminated forensic DNA samples, thus increasing by manifold the probability of obtaining STR profiles for forensic HID purposes.

4 Concluding remarks:

STR-capture or enrichment technique developed in this study was able to address the two major challenges encountered by forensic community by presenting a quick, reliable and cost effective methodology. The method provided an increased tolerance to non-human DNA contamination even up to 5 folds and against various potential PCR inhibitors by 3-4 folds. Further, the enrichment method can be applied with initial DNA concentrations of as low as 2 ng and also compatible with widely used commercial kits (AmpFlSTR® Identifiler® PCR amplification kit and Powerplex® 16 HS system) to continue with the downstream amplification process in a multiplex fashion. This technique can be further refined by increasing the number of target loci to include all the additional forensic STRs like those mentioned in GlobalfilerTM (3) and Promega Powerplex® Fusion systems (http://au.promega.com/resources/profiles-in-dna/2013/evaluation-of-the-powerplex-fusion-system-for-use-on-the-abi-prism-310-genetic-analyzer/). It can also be considered for automation to apply routinely on compromised forensic specimens. Moreover, this technique can be exploited to capture the limiting amounts of desired target regions in other fields such as medical genetics and evolutionary genetics as well wherein the template DNA molecules are either limiting or mixed with contaminants which inhibit downstream assay reactions and further improvements can be carried out for higher multiplexing in large-scale studies.


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  1. List of 18 pairs of biotinylated oligos

S.No.

Locus

5’- biotinylated oligonucleotides (5’→ 3’)

1

FGA

F

GAA GTA GCT GCT GAG TGA TTT GTC TGT AAT TG

R

GGT TTT GAA CTC ACA GAT TAA ACT GTA ACC A

2

D18S51

F

ACT GCA CTT CAC TCT GAG TGA CAA ATT GAG ACC T

R

GTG GAG ATG TCT TAC AAT AAC AGT TGC TAC

3

THO1

F

TTC CGA GTG CAG GTC ACA GGG AAC ACA GAC T

R

GGG GTG ATT CCC ATT GGC CTG TTC CT

4

CSF1PO

F

GCA TTT CCT GTG TCA GAC CCT GTT CTA AGT AC

R

CCC TGT GTC TCA GTT TTC CTA CCT GTA A

5

D7S820

F

ACG ATT CCA CAT TTA TCC TCA TTG ACA GAA TT

R

AAG GGT ATG ATA GAA CAC TTG TCA TAG TTT AGA AC

6

D13S317

F

ACT CTC TGG ACT CTG ACC CAT CTA ACG CCT A

R

CCA TAG GCA GCC CAA AAA GAC AGA CAG

7

D8S1179

F

ACT TAT ATG TAT TTT TGT ATT TCA TGT GTA CAT TCG

R

TAC CTA TCC TGT AGA TTA TTT TCA CTG TGG

8

D3S1358

F

TCA TGA AAT CAA CAG AGG CTT GCA TGT ATC TA

R

AGT CCA ATC TGG GTG ACA GAG CAA GA

9

D2S1338

F

AGC CAG TGG ATT TGG AAA CAG AAA TGG CTT GG

R

GTA CCT GCA GGT GGC CCA TAA TCA TG

10

D19S433

F

ACT GCA CTC CAG CCT GGG CAA CAG AAT AAG AT

R

GAT ATT TTG GTG CAC CCA TTA CCC GAA T

11

VWA

F

AGC CCT AGT GGA TGA TAA GAA TAA TCA GTA TG

R

GGA CAG ATG ATA AAT ACA TAG GAT GGA TGG

12

AMEL

F

GGC CAA CCA TCA GAG CTT AAA CTG GGA AGC TG

R

TCC CCT GGG CTC TGT AAA GAA TAG TGG

13

TPOX

F

TGG CAC AGA ACA GGC ACT TAG GGA ACC CTC AC

R

GGCC CTT CTG TCC TTG TCA GCG TTT ATT

14

D16S539

F

AAG CTC TTC CTC TTC CCT AGA TCA ATA CAG ACA G

R

GTT TGT GTG TGC ATC TGT AAG CAT GTA TC

15

D5S818

F

TCA TAG CCA CAG TTT ACA ACA TTT GTA TCT TTA T

R

GAC AAG GGT GAT TTT CCT CTT TGG TAT CC

16

D21S11

F

AAA TAT GTG AGT CAA TTC CCC AAG TGA ATT GC

R

GTA TTA GTC AAT GTT CTC CAG AGA CAG AC

17

Penta D

F

TCC AGC CTA GGT GAC AGA GCA AGA CAC CAT

R

GC CTA ACC TAT GGT CAT AAC GAT TTT TTT GAG

18

Penta E

F

TTT GGG TTA TTA ATT GAG AAA ACT CCT TAC AAT TT

R

TTG AAC CCA GGA GGT GGA GAT TGA AGT G

  1. Tolerance limits of AmpFlSTR® Identifiler® plus PCR amplification kit with and without enrichment

Inhibitor

Tolerance limit without enrichment (amplification by AmpFlSTR® Identifiler® plus kit)

Tolerance limit with enrichment (amplification by AmpFlSTR® Identifiler® plus kit)

Hematin

0.3 mM

1 mM

Humic acid

0.15 µg/µl

0.4 µg/µl

Tannic acid

0.3 µg/µl

1 µg/µl

Calcium

1 mM

4 mM

  1. Number of loci obtained without and with modified PEC-based enrichment of STR loci from forensic samples.

Sample

qPCR estimation of the DNA concentration (ng/ µl)

Number of loci amplified without enrichment (out of 16)

Number of loci amplified after enrichment (out of 16)

1

3.36

13

16

2

0.11

0

14

3

-

9

13

4

0.46

11

13

5

1

0

3

6

1.054

8

10

7

-

0

0

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2

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3

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4

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