P-001
Complex Paternity
investigations: The need for more genetical information
Abrantes D1, Pontes ML1,
Lima G1, Cainé L1, Pereira MJ1, Matos P1,
Pinheiro MF1,2
1Instituto
Nacional de Medicina Legal – Delegação do Porto
2Faculdade
de Ciências da Saúde – Universidade Fernando Pessoa
In
the past few years our laboratory has been registering a raising demand of
difficult parentage investigations, namely those performed in the absence of
the putative father’s genetical data. Due to the also increasing complexity of
the aforementioned investigation cases themselves, the results provided by the
routinely used commercial kits AmpFℓSTR® Identifiler™ (Applied Biosystems) and Powerplex® 16 (Promega), in our laboratory, even in
conjunction with the complementary commercial kit Powerplex® ES Monoplex System (SE33), are no longer
always satisfactory. Therefore, there is an urgent need for more (preferably)
easily- and rapidly-analysable markers. In this sense, our laboratory resorted
to the long-time commercialized kit Gene Print® Fluorescent STR Systems FFFL Multiplex
(Promega), which allows for the co-amplification of four more STR-loci (F13A01, FES/FPS, F13B e LPL) and,
thus, for the acquisition of the required supplementary genetical data. In this
work, we describe several cases to whose solution the FFFL data were crucial.
We also report the allelic frequencies and some parameters of forensic
interest, relative to FFFL loci, for
the Northern Portuguese population.
Contact: Biologia@dpinml.mj.pt
P-002
Accurate mtDNA mixture quantification
using the Pyrosequencing technology
Allen M, Nilsson M, Andréasson H
Department of Genetics and
Pathology, Rudbeck Laboratory, Uppsala
University, Sweden
Analysis of mtDNA variation using Sanger sequencing
does not allow accurate quantification of mtDNA mixtures. Thus, a method to
determine the specific mixture ratios in samples displaying heteroplasmy,
consisting of DNA contribution from several individuals or containing
contamination would be valuable. In this study, a novel quantification system
for mtDNA mixture analysis is described. The assay is based on pyrosequencing
technology, in which the linear relationship between incorporated nucleotides
and released light allows accurate quantification. The routinely applied Sanger
sequence analysis of mtDNA is robust and in most cases successful due to the
high copy number of mtDNA per cell. However, occasionally samples show a DNA
mixture as a consequence of multiple contributors, heteroplasmy or
contamination. In contrast to STR analysis, quantification of mixed samples
based on mtDNA sequence analysis is not feasible using the current sequencing
methodology. The ABI PRISMÒ BigDyeTM
Primer Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA) is commonly used for
mtDNA analysis. Although this chemistry easily and effectively determines the
sequence in single source samples, the uneven peak heights and
sequence-dependent variation in dideoxynucleotide incorporation efficiency
prevent quantification of mixtures. Thus mixtures can be detected and
visualised using Sanger sequencing, but the information cannot be used to
determine the exact quantities of the different mitochondrial types and in most
cases the results are called as inconclusive. Resolution of mtDNA mixtures has
been demonstrated previously using alternative technologies such as cloning and
denaturing high-performance liquid chromatography. However, in order to resolve
and accurately quantify multiple individuals contributing to a sample in equal
or unequal ratios, an easy to use quantitative assay would be useful.
Pyrosequencing is a technology based on the release of pyrophosphate during
strand elongation, producing light. The light signal is proportional to
incorporated nucleotides, allowing allele quantification utilising PSQTM96MA
SNP Software (Version 2.02, Biotage, Uppsala,
Sweden). The
allele quantification capability has been previously used in a number of
studies, including allele frequency measurements in pooled DNA samples and
quantitative analysis of methylation status at CpG islands. Other studies
involve gene copy number measurements and determination of allele-specific
transcript expression. In this study, a subset of PCR fragments previously
developed for a fast and simple pyrosequencing analysis of variation in the
mtDNA control and coding region, were used for pyrosequencing based
quantification. Seven polymorphic sites, three in the control region and four
in the coding region, within five PCR fragments, were selected and successfully
used for mtDNA mixture quantification. For all SNPs quantified in this study, a linear
relationship was observed between measured and expected mixture ratios. The average standard
deviations of each of the seven SNPs fell within the expected 1-2% (for 10
replicate reactions). In conclusion, this mtDNA
mixture quantification system is an alternative application of the
pyrosequencing technology and is useful in forensic DNA analysis.
Pyrosequencing has been shown to provide a very rapid, accurate and easy to use
quantification system that can be used in forensic casework investigations to
resolve and interpret major and minor mtDNA contribution from multiple
individuals, determine heteroplasmy ratios and monitor contamination.marie.allen@genpat.uu.se
P-003
Reducing mtDNA
sequencing efforts by half in forensic casework
Allen M, Nilsson M, Calloway C, Divne A-M
Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Sweden
Mitochondrial DNA (mtDNA)
sequencing can be time-consuming and laborious, limitations that can be
minimized using a faster typing assay. The LINEAR ARRAY mtDNA HVI/HVII
Region-Sequence Typing Kit (Roche Applied Science) uses sequence-specific
probes immobilized in 31 lines. Linear array typing of mtDNA polymorphisms is a
simple and fast pre-screening method with potential to substantially reduce
sequencing efforts due to exclusion of samples. The analysis is performed in
less than 3 hours without the need for expensive equipment.
Over a five-year period more
than 300 forensic samples have been successfully typed for mtDNA polymorphisms
using these linear arrays in our laboratory. A majority of the specimens that
were analyzed using the combined HVI/HVII linear array were shed hairs. However,
previous use of the HVII linear array has been successful on samples obtained
from a variety of items, such as epithelial cells collected from areas in close
contact with the skin. Furthermore, successful linear HVI/HVII array typing
results have been obtained in a cold case investigation of 15-years old hair
samples, one shed head hair (3 cm long) and two reference hairs.
A high sensitivity in the
assay was shown by typing of TaqMan quantified DNA samples with limited
amounts. Successful and reliable results were obtained from three centimetre
pieces of distal shaft parts of shed and plucked hairs. Moreover, strong and
easily interpretable array signals were obtained from control samples
containing 100 -10 000 mtDNA copies, equivalent to 0.6 pg to 60 pg of genomic
DNA (333 genome equivalents/ng DNA) indicating a highly sensitive typing
system.
The exclusion capacity has
been evaluated by a retrospective study of 90 previously HVI/HVII sequenced
samples (57 evidence samples and 33 reference samples) from 16 forensic cases.
Using the HVI/HVII mtDNA linear array, 56% of the samples were excluded and
thus less than half of the samples require further sequencing due to a match or
inconclusive results. Of all the samples that were excluded by sequence analysis,
79% could be excluded using the HVI/HVII linear array alone.
The use of the mtDNA linear
arrays in our laboratory has served as a valuable pre-screening method and
demonstrates the potential to reduce the required sequencing efforts by more
than half. Thus, this rapid and user-friendly linear array typing system
provides a convenient and efficient pre-screening method for selection of the
samples of most interest for further investigation.
P-004
The
Amelogenin locus displays a high frequency of X homologue failures in São Tomé
island (West Africa)
Alves C1, Coelho M1, Rocha J1,2,
Amorim A1,2
1
IPATIMUP, R. Dr. Roberto Frias, s/n, 4200-465 Porto, Portugal
2
Faculdade de Ciências da Universidade do Porto, Pr. Gomes Teixeira, 4009-002 Porto, Portugal
A multiplex STR study using
the Powerplex 16 System (Promega) in 503 unrelated individuals from the island of São Tomé (Gulf of Guinea,
West Africa) revealed 10 male individuals
presenting only the Y homologue of the Amelogenin locus (~2%). These
individuals were further typed with other commercial kits which also amplify
the Amelogenin locus, namely the AmpFLSTR Identifler (Applied Biosystems) and
Y-PlexTM12 (Reliagene) kits, and an X/Y genotype was only obtained
with the primers used in Y-PlexTM12.
Although this X Amelogenin
drop-out was only detected in males, this does not rule out the fact that
females may also carry it. Since women have two X chromosomes, in some instances
it could be suspected that an X failure was also present in females, by simple
observation of differences in electrophoregram peak heights, in comparison with
XY profiles. Although we cannot objectively consider these apparent nulls in
females for frequency estimate purposes, there is no doubt of its magnitude in
this population. With a 2% frequency in males, it is expected that the
frequency of female carriers and homozygotes will be 3.92% and 0.04%,
respectively. It is also noteworthy that the previously reported frequency for
X Amelogenin null (which was estimated in Caucasians) was much lower (0.3%).
Failure to amplify alleles in
the Amelogenin locus has been described before, mainly in cases where the Y
homologue fails, which can have critical consequences in forensic casework.
Cases where the X counterpart fails to amplify, as described here, are not of
fundamental significance in forensic genetics, since there is no danger of a
male individual being mistaken for a female one. However, this can have a
different impact in other fields, such as in prenatal diagnosis of certain XY
chromosome abnormalities, like XXY, using quantitative assays.
The high frequency of
amplification failures already detected for either the X or Y chromosome
Amelogenin locus, only draws our attention more to the need for caution when
applying solely the amelogenin test for sex determination.
Sequencing of the X Amelogenin
allele responsible for the amplification failure in the 10 male individuals is
undergoing.
P-005
Making
the most of Y-STR haplotypes. The HapYDive
Alves C1, Gusmão L1, Meirinhos J1,
Amorim A1,2
1
IPATIMUP, R. Dr. Roberto Frias, s/n, 4200-465 Porto, Portugal; 2
Faculdade de Ciências da Universidade do Porto, Pr. Gomes Teixeira, 4009-002 Porto, Portugal
Since the informative power of
a Y-STR marker can only be recognised in a haplotype context, a software was
devised to evaluate the increase of haplotype diversity (HD) by the addition of
any combination of markers to a fixed number of markers. The first version of the
program was quite limited and not very user-friendly. The HapYDive is the
latest version, created in Excel format (available at www.ipatimup.pt/app/). It´s not only a
software for Y-STR HD calculation but, more importantly, it allows the
determination of which combination of Y-STRs is the most informative in a
certain population sample. With the HapYDive it is possible to analyse any set
of Y markers up to a maximum of 20, with a minimum number of 4 markers fixed
for calculations.
As an example, let’s consider the fixed set of Y-STRs
currently used in the "YHRD - Y Chromosome Haplotype Reference
Database" (http://www.yhrd.org), comprising the “minimal haplotype”
markers (9 loci) plus DYS438 and DYS439. Depending on the population sample,
these 11 loci together will have a certain HD value. Which set and what number
of the other available Y-STRs will increase more rapidly the HD value?
Applying the HapYDive program to a population sample
from Portugal (N=657) with haplotypes containing the 11 Y-STRs plus DYS437,
DYS460, DYS461, DYS635, GATA A10 and GATA H4, the best order of markers is
shown in Table 1. In this sample, all the other Y-STRs contribute to a certain
degree to an increment of HD, but DYS460 contributes the most and DYS635 the
least.
However, in other samples, particularly in those from
different population groups, one or more Y-STRs may not contribute in any way,
and the order in which they´ll contribute more may be quite different. For
example, by applying the HapYDive to a population sample from Mozambique
(N=112) using the same markers, the best order is shown in Table 2. In this
case, the order is different and there is one marker, DYS437, that does not
contribute in any way to a higher HD.
Apart from applying this program to different sample
origins and to different sets of Y-STR markers (namely from the recent
commercial kit Yfiler from Applied Biosystems), it is also worth studying the
effect of sample size. This study is still undergoing and a discussion of the
results will be shown. Contact: calves@ipatimup.pt
|
Table 1 (Portugal)
|
|
|
Table 2 (Mozambique)
|
|
|
Y-STR sets
|
HD
|
|
Y-STR sets
|
HD
|
|
11 Y-STRs
|
0.99771
|
|
11 Y-STRs
|
0.99212
|
|
11 Y-STRs + DYS460
|
0.99866
|
|
11 Y-STRs + GATA A10
|
0.99437
|
|
12 Y-STRs + GATA H4
|
0.99915
|
|
12 Y-STRs + DYS460
|
0.99582
|
|
13 Y-STRs + GATA A10
|
0.99936
|
|
13 Y-STRs + DYS635
|
0.99646
|
|
14 Y-STRs + DYS461
|
0.99950
|
|
14 Y-STRs + DYS461
|
0.99695
|
|
15 Y-STRs + DYS437
|
0.99960
|
|
15 Y-STRs + GATA H4
|
0.99727
|
|
16 Y-STRs + DYS635
|
0.99966
|
|
16 Y-STRs + DYS437
|
0.99727
|
P-006
Estimating the
post-mortem interval (I)
The use of genetic
markers to aid in identification of Dipteran species and subpopulations
Ames C, Turner B, Daniel B
Department of Forensic Science
and Drug Monitoring, King’s College London, UK
Insect
evidence can be utilised in a forensic investigation in a variety of ways. For
instance, insects are most commonly used to help in the estimation of time
since death of a discovered corpse. With a knowledge of recent environmental
conditions of the scene of crime, an entomologist can predict how long it has
taken for any insects present to have reached their particular developmental
stage and hence the minimum time since death. The insect species present on a
corpse will also indicate the time since death as insect species colonise a
carcass in a distinct succession. Some insects have distinct geographical
distributions, their presence outside of their normal habitat could indicate
post mortem movement of a corpse or link a suspect to a scene of crime. Their
lifecycles are seasonal and presence of insects can therefore indicate the time
of year a crime occurred. The presence of drugs or poisons within feeding
insects may give an indication of cause of death of a body.
All forensic entomology
techniques depend upon accurate identification of insect species. At present
this is mainly based upon morphological differences between species. This can
be difficult as the early lifecycle stages of many forensically important
Dipteran species are very hard to distinguish.
One
aim of this work was to use DNA molecular markers to help in identification of
forensically important fly species and ultimately populations within the UK.
Wild
populations of Calliphora vicina and Calliphora vomitoria
(Diptera: Calliphoridae) caught from various locations around the UK were raised
and maintained in the laboratory. Both these species are early corpse invaders
in the United Kingdom.
To ensure the identity of both species before experimental work began, they
were characterised morphologically using a key (Smith 1986). DNA was extracted
from adults and larval forms. Regions of both the nuclear (xanthine dehydrogenase
exon 2) and mitochondrial (cytochrome oxidase I and the control region) genomes
were amplified using PCR and then sequenced or digested using restriction
enzymes.
These
molecular markers have been shown to contain both interspecific and intra specific
variation and thus can be used to distinguish between the two species and also
between English populations of both species.
Contact:
carole.ames@kcl.ac.uk
P-007
Estimating
the post-mortem interval (II) The use of differential temporal gene expression
to determine the age of blowfly pupae
Ames C, Turner B, Daniel B
Department of Forensic Science
and Drug Monitoring, King’s College London, UK
Insect
evidence can be utilised in a forensic investigation in a variety of ways. For
instance, insects are most commonly used to help in the estimation of time
since death of a discovered corpse. With a knowledge of recent environmental
conditions of the scene of crime, an entomologist can predict how long it has
taken for any insects present to have reached their particular developmental
stage and hence the minimum time since death. The insect species present on a
corpse will also indicate the time since death as insect species colonise a
carcass in a distinct succession. Some insects have distinct geographical
distributions, their presence outside of their normal habitat could indicate
post mortem movement of a corpse or link a suspect to a scene of crime. Their
lifecycles are seasonal and presence of insects can therefore indicate the time
of year a crime occurred. The presence of drugs or poisons within feeding
insects may give an indication of cause of death of a body.
To
establish time since death, an entomologist requires accurate assessment of the
age of insects discovered associated with a corpse. At present this is done
using morphological features or biometric characteristics such as length or
weight. The aim of this work was to use molecular techniques to determine the
age of immature forms of forensically important fly species. Throughout the
developmental lifecycle of insects different genes will be expressed at
specific time points. Once identified these temporally expressed genes could
provide markers as to the age of an insect.
Wild
populations of Calliphora vicina (Diptera: Calliphoridae) were maintained
in the laboratory. This species is an early corpse invader in the United Kingdom.
Initially the pupal stage would be focussed upon. Adult females were encouraged
to lay eggs and this was taken as ‘time zero’. Eggs were placed at a constant
20C until the pupal stage. At specific timepoints
total RNA was extracted from pupal samples. The extracted mRNA was reverse
transcribed to cDNA. Potential markers were located either from the use of
differential display techniques (DD) or from the literature. DD is a method
that detects changes in gene expression between samples by the random
amplification of cDNA. Fragments are visualised on a gel and differences in
banding pattern can be focussed upon.
Once
potential markers were located their expression in differently aged pupal
samples was quantified using Real-time PCR. The results indicated that this is
a viable method for age determination of Dipteran immature stages.
Contact:
carole.ames@kcl.ac.uk
P-008
Extended
Northern Portuguese database on 21 autosomal STRs used in genetic
identification
Amorim A1,2, Alves C1, Gusmão L1,
Pereira L1
1IPATIMUP,
R. Dr. Roberto Frias, s/n, 4200-465 Porto, Portugal;2Faculdade de
Ciências da Universidade do Porto, Pr. Gomes Teixeira,
4009-002 Porto, Portugal
Routine casework in our genetic
identification laboratory is carried out with two commercially available
autosomal STR kits, namely Identifiler (Applied Biosystems) and Powerplex 16
(Promega), which together amplify a total of 17 STR loci. In some instances,
namely in deficient paternity cases, we can also count on an in-house multiplex
system which amplifies 4 more loci, totalling 21 STRs. Along the years, we have
accumulated population frequency data in our database, namely from individuals
residing in Northern Portugal, which we are now presenting extensively,
together with parameters of forensic interest. This is also the first time we
are presenting data on both D2S1338 and D19S433. The following table summarises
our results:
|
|
CD4
(N=382)
|
CSF1PO
(N=1825)
|
D2S1338
(N=760)
|
D3S1358
(N=1816)
|
D5S818
(N=1816)
|
D7S820
(N=1809)
|
D8S1179
(N=1822)
|
|
Ho
|
0.696
|
0.731
|
0.833
|
0.784
|
0.702
|
0.794
|
0.808
|
|
He
|
0.705
|
0.724
|
0.877
|
0.787
|
0.702
|
0.810
|
0.811
|
|
PD
|
0.855
|
0.872
|
0.973
|
0.921
|
0.859
|
0.937
|
0.941
|
|
CE
|
0.422
|
0.452
|
0.741
|
0.556
|
0.436
|
0.601
|
0.616
|
|
P
|
0.929
|
0.496
|
0.798
|
0.754
|
0.597
|
0.112
|
0.034
|
|
|
D13S317
(N=1818)
|
D16S539
(N=1283)
|
D18S51
(N=1810)
|
D19S433
(N=761)
|
D21S11
(N=1817)
|
F13A01
(N=484)
|
FES
(N=487)
|
|
Ho
|
0.773
|
0.760
|
0.891
|
0.806
|
0.843
|
0.711
|
0.700
|
|
He
|
0.782
|
0.776
|
0.876
|
0.794
|
0.843
|
0.754
|
0.700
|
|
PD
|
0.922
|
0.915
|
0.972
|
0.930
|
0.957
|
0.899
|
0.852
|
|
CE
|
0.566
|
0.546
|
0.733
|
0.587
|
0.673
|
0.509
|
0.417
|
|
P
|
0.495
|
0.760
|
0.919
|
0.820
|
0.552
|
0.098
|
0.818
|
|
|
FGA
(N=1833)
|
MBPB
(N=371)
|
Penta
D
(N=1280)
|
Penta
E
(N=1281)
|
TH01
(N=2403)
|
TPO
(N=2402)
|
VWA
(N=2303)
|
|
Ho
|
0.857
|
0.741
|
0.837
|
0.899
|
0.783
|
0.639
|
0.804
|
|
He
|
0.866
|
0.728
|
0.839
|
0.885
|
0.796
|
0.648
|
0.810
|
|
PD
|
0.967
|
0.879
|
0.953
|
0.976
|
0.927
|
0.823
|
0.937
|
|
CE
|
0.712
|
0.469
|
0.657
|
0.755
|
0.570
|
0.386
|
0.602
|
|
P
|
0.316
|
0.360
|
0.015
|
0.786
|
0.658
|
0.691
|
0.915
|
N: nº
individuals; Ho: observed heterozygosity; He: expected heterozygosity according
to Nei; PD: power of discrimination; CE: a priori chance of exclusion; P:
Hardy-Weinberg equilibrium, exact test based on more than 2000 shufflings, for
standard error <0.01.
Deviations from Hardy-Weinberg equilibrium were
detected in D8S1179 and Penta D loci, but applying the Bonferroni correction
for the number of loci analysed, the departure in both loci was not significant
(0.05/21=0.0024).
Both commercial STR kits share 13 loci but use
different primer pairs, and so genotype inconsistencies may occur. For individuals
genotyped as homozygotes with one kit and as heterozygotes with the other, the
latter genotype was the one considered.
The overall matching probability for the 21 STRs in
our population sample is of 1 in 1.56 x 1024 individuals, and
combined power of exclusion of 0.9999999914.
P-009
Evaluation of
the 11 Y-STR loci in the PowerPlex® Y-system;
Experience from
analyses of single male samples and simple male: male mixtures.
Andreassen R, Heitman IK
Institute of Forensic Medicine, University of Oslo,
Norway
Interpretation of sample mixtures requires knowledge
about the efficiency of the system. In this study we have measured the amount
of stutter and pull-up in 100 male samples analysed at 11 Y-STR loci (DYS 391,
DYS389I/II, DYS 439, DYS 393, DYS 390, DYS 385, DYS 438, DYS 437, DYS 19, DYS
392) using the PowerPlex® Y-system as described by the manufacturer.
The proportion of stutter differed among the loci in the multiplex-kit and was
associated with allele length (number of repeats) and repeat size (3-5
basepairs). The best performing locus was DYS 438 while largest proportion of
stutter was observed in locus DYS 389II. Area of stutter was less than 0.2 for
all loci tested. Both stutter in position N-2 and N+1 was observed at certain
loci. No pull up larger than 0.1 was observed in the loci analysed. Locus DYS
393 was amplified less efficient than other loci in the multiplex mix giving
alleles with low peak heights compared to the others. Efficiency for each Y-STR
locus will be presented in detail. Fourteen samples from males with known
Y-haplotypes were used to compose male:male mixtures. Mixture ratios varied
within the interval 1:3 to 1:1. A total of sixty-five samples were analysed. In
each sample the peak areas of alleles were used to type a “minor” and a “major”
Y-haplotype consisting of all minor alleles or major alleles, respectively, at
loci with two alleles. The results from this exercise were compared with the
two known Y-haplotypes in each sample. Disregarding locus DYS 393 and DYS 385
a/b, the two Y-haplotypes in a sample was correctly typed in all samples with a
relative peak area difference (average of peak areas of minor alleles / peak
area of major alleles) less than 0.6. The results from this simple test
indicate that peak area of alleles in PowerPlex® Y-system provides
quantitative information that might be used to interpret the most likely
Y-haplotypes in a simple male:male mixture.
P-010
Icelandic
population data for the 10 autosomal STR loci in the AMPFlSTR®SGM Plus™ system and the 11 Y-STR loci in the
PowerPlex® Y-system
Andreassen R, Heitman IK,
Hansen L, Mevaag B
Institute of Forensic Medicine, University of Oslo,
Norway
Autosomal STR
polymorphisms at 10 loci (D3S1358, vWA, D16S539, D21S1338, D8S1179, D21S11,
D18S51, D19S433, TH01 and FGA) and Y-STR polymorphisms at 11 loci (DYS 391,
DYS389I/II, DYS 439, DYS 393, DYS 390, DYS 385, DYS 438, DYS 437, DYS 19, DYS
392) are presented. Samples from a population material of unrelated individuals
from Iceland
were analysed using the AMPFlSTR®SGM
Plus™ system (n=110) and the PowerPlex® Y-system (n=76) as described
by the manufacturer. For the autosomal polymorphisms the observed
heterozygosities ranged from 0,764 (vWA) to 0,891 (FGA). No significant
deviation from Hardy-Weinberg equilibrium was observed. For the Y-chromosome
polymorphisms 62 different haplotypes were observed in the 76 male samples
analysed. No haplotype was observed more than three times in the population
sample.
Locus
diversity, allele distributions and other relevant forensic genetic parameters
will be presented in detail.
P-011
Low
Copy Number: interpretation of evidence results
Anjos MJ1, Andrade L1, Carvalho
M1, Lopes V1, Serra A1, Oliveira C1,
Balsa F1, Brito P1, Corte-Real F2, Vide MC1
1 Forensic Genetic Service. National Institute of Legal
Medicine. Largo da Sé Nova, 300 Coimbra. Portugal
2
National Institute of Legal Medicine. Largo da Sé Nova, 300 Coimbra. Portugal
geneforense@dcinml.mj.pt
Evidence
of crime scene and samples from decomposed or skeletal remains have in many
cases, low amounts or degraded DNA.
The
choice of extraction protocols as well as sensitive and robust STR’s is crucial
to obtain good results but in many situations is not enough; it is necessary to
make protocol adaptations of amplification instructions even when are used
commercial kits.
One
of the most common modifications is changing the number of cycles in the
amplification protocol, with an increment of 4, 6 or more cycles. However in
some cases there are difficulties in interpreting results because extra peaks
appear or an imbalance between them seems to have no sense.
In
this work we show some cases where changes of technical approaches produced
better results and others where we had problems in interpreting evidence
results.
P-012
Amelogenin as a Target for Real Time PCR Quantitation of Forensic Templates
Anwar N, Goodman M, Hulme P,
Elsmore P, Greenhalgh M and McKeown B
Orchid
BioSciences (Europe) Ltd, Abingdon, UK
PCR
is the ubiquitous method of forensic DNA analysis, but prior to amplification,
two other processes are crucial to obtaining a satisfactory result: template
DNA extraction and template quantitation.
Here we will presume the extraction process has been performed, and
concentrate on the quantitation step: a focus of recent advances. Real time quantitative PCR (RT-QPCR) is an
advantageous alternative to probe hybridisation or fluorescent dye association,
which are (respectively) laborious and less accurate procedures. We reviewed the available commercial methods
of RT-QPCR and concluded that for our requirements, a more attractive solution
was the in-house development and validation of an ultra-rapid, small batch size
solution. Our solution is real time detection using the Roche
LightCycler 2.0 and the amplification of a 106/112bp amelogenin amplicon. Melt-curve analysis and back extrapolation to
the starting template-dependant crossing point generates results from 32
samples in ~30 minutes (post PCR assembly) and this approach has advantages in
that a positive quantitation result implies that in the SGM Plusä
amplification that follows, at the very least, an amelogenin product should be
generated. The use of the amelogenin
target also provides an indication of the possibility of PCR product travelling
from the separate PCR product room backwards into the clean PCR set-up
environment, something that the use of telomerase or b-globin
amplicons cannot provide. We have
validated the use of our LightCycler-amelogenin based quantitation system and
have seen significant improvements in the reliability of quantitation
measurements in our forensic laboratories.
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