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The Alu family of short interspersed repeated DNA elements are distributed
throughout primate genomes. They are the most abundant transponons in the
human genome, accounting for 10% of its mass. It has previously been established
that Alu polymorphisms TPA-25, Angiotensin Converting Enzyme (ACE), YaNBC51a
and YaNBC182 are a useful indicators of genetic variation between individuals and
populations and thus are beneficial in forensic analysis.
The aim of this study is to screen the Forensics DNA analysis class 2008, along with
2 unknown samples, for the insertion/deletion of Alu polymorphisms and to compare
this with external populations. The Alu polymorphisms used are ACE, TPA-25,
YaNBC51, YaNBC182 and AluSTYa.
The Chelex method was used to extract DNA, which was then amplified by PCR and
viewed on a transilluminator. Analysis included calculations of allele and genotype
frequencies, heterzygosities, match probabilities and discriminative probabilities,
profile frequencies and likelihood ratios, and an evaluation of the Hardy-Weinberg
Equilibrium (HWE).
In the class population only ACE polymorphism did not exist in HWE, whereas all
markers in the pooled population were in HWE. Both class and pooled data had a
discrimination power of 0.98 which concludes that there is a potential power of 98%
to discriminate between two individuals chosen at random. Sample 39, with a profile
frequency of 0.026 was the most common with a likelihood ratio of 39 to 1. This
concludes that within our class a higher percentage of people will have a similar
profile frequency as sample 39.
Alus are found to be useful in forensic analysis as they are less laborious and
therefore cheaper to use, they have a low mutation making them a unique event
polymorphism.
Introduction
In forensic analysis, Alu polymorphisms are preferred over VNTRs and RFLPs as
unlike the latter two, Alus are PCR able. The low mutation rate of Alus makes them
unique event polymorphisms and robust markers, making them useful in forensics
analysis (Gasper, 2004). They are also less laborious and cheaper to use than other
polymorphisms.
The most commonly studied Alus areTPA-25 and ACE, both of which are human
specific (Batzer et al. 1996) and belong to the Ya5/8 subfamilies (Roy et al. 2000).
As they have retroposed relatively recently they have not yet been fixed at specific
loci on chromosomes (Batzer et al. 1994).
Aims
Method
Subjects
The study compromised of 67 undergraduate students from the 2008 Forensic DNA
analysis class at Loughborough University. Each individual was given an ID number,
which was used to label the 1.5ml microfuge tubes. 1 ml of RNAse/DNAse free water
was added into each the microfuge tube using a pipette.
Individuals wiped the inside of their cheek with a cytology cotton swab to dislodge
loose cells. The swab was then rotated vigorously in the microfuge tube, and stirred
periodically for 30 minutes at room temperature to free collected cells. Two 4 x 3mm
diameter punches of the bloodstained filter paper were provided (unknown samples
-see Appendix1).
DNA from collected cells and unknown samples were successfully extracted using
the Chelex method (Appendix 2).
Maximum amount of supernatant was extracted from each sample without disturbing
pellet/filter paper and then transferred into a clean labelled centrifuge tube. 0.2ul
PCR tubes were labelled with ID numbers and corresponding master mix ID. 20µl of
relevant master mix was transferred to the labelled PCR tubes. 5µl of the extracted
DNA were added and gently mixed together.
Genotyping
The samples were then amplified using multiplex PCR, which uses more than one
set of primers in one reaction. Reactions were carried out using the appropriate
mastermixes containing specific primers for the Alu polymorphism being analysed
(Appendix 3). (Mastermixes for PCR reactions can be found in Appendix 4). Two
duplex PCR reactions were used for autosomal loci (one for Alu polymorphisms
3
Products of PCR were then separated using gel electrophoresis using a 2% agarose
gel set in a mould containing wells (preparation shown in Appendix 6). Combs were
put in place, and Ethidium Bromide TBE was poured into the electrophoresis tank
covering the gel in 1-2 mm of solution. After the combs were removed, a reference
sample containing the investigated alleles with known fragment sizes was inserted,
creating a ladder to allow for identification of band sizes. 10µl of samples were then
inserted into the wells and electrophoresed for 30 minutes at 100volts. Gels were
then viewed on a transilluminator to visualise the bands.
Materials- 20% Chelex, PCR Master Mix, RNAse/ DNAse free water, Control
Sample, Agarose, Ethidium Bromide/SYBR Safe Dye, Tris Boric EDTA (TBE) buffer
pH8.3
Statistical Analysis
For each polymorphism, allele frequencies were calculated using gene counting
method and are shown with standard errors. Chi square statistics were used to
consider deviations from Hardy-Weinberg. To analyse allele diversity of the class,
observed and expected heterozygosities were calculated. Random match
probabilities were calculated for each database along with discriminative probability,
and compared to the pooled data. To determine the probability of the genotypes
occurring in the pooled and class database, profile frequencies and likelihood ratios
were calculated for the individual samples within our group and our unknown
samples (samples 36-41). However the unknown samples were not included in the
databases constructed as they were unrepresentative of the class population.
Results
Participant L 36 37 38 39 40 41
No (unknown) (unknown)
ACE DD II ID ID ID II
TPA25 II DD DD DD DD ID
Participant L 36 37 38 39 40 41
No (unknown) (unknown)
182 ID ID - ID - DD
51A DD DD - II - DD
Participant L 36 37 38 39 40 41
No (unknown) (unknown)
Gender F F F F F M
Figures 1-3 show electrophoresis images which show the band detection of the
polymorphisms of four participants and two unknown samples. The table beneath
each displays the genotypes observed in relation to the ladder (L).
II ID DD II ID DD
Genotype frequency
0.172 0.25 0.578 0.211 0.482 0.307
I D I D
Allele frequency
0.297 0.703 0.452 0.548
Standard Error 0.04 0.04 0.033 0.033
In Hardy-Weinberg
X2=10.3 X2=0.077
Equilibrium
Df=1, CV=3.84 Not HWE In HWE
Observed Heterozygosity 0.25 0.482
Expected Heterozygosity 0.417 0.495
Match Probability 0.426 0.371
Probability of Discrimination 0.574 0.629
II ID DD II ID DD
Genotype frequency
0.3 0.383 0.317 0.269 0.433 0.299
Allele frequency I D I D
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II ID DD II ID DD
Genotype frequency
0.193 0.526 0.281 0.185 0.481 0.333
I D I D
Allele frequency
0.456 0.544 0.426 0.574
Standard Error 0.047 0.047 0.03 0.03
In Hardy-Weinberg
X2=0.211 X2=0.032
Equilibrium
Df=1, CV=3.84 In HWE In HWE
Observed Heterozygosity 0.526 0.481
Expected Heterozygosity 0.496 0.489
Match Probability 0.393 0.377
Probability of Discrimination 0.607 0.623
Table 5 – Combined Match Probability and Discrimination of Class and Pooled data
Class Pooled
Combined Match Probability 0.021 0.017
Combined Probability of 0.979 0.983
Discrimination Value
% discriminatory 97.90% 98.30%
Table 6 - Profile Frequency and Likelihood ratio of class and pooled data.
Likelihood
Ratio(Class) 289.428 58.142 4.049 38.932 4.049 133.191
Profile Freq
(Pooled) 0.011 0.009 0.148 0.013 0.148 0.008
Likelihood Ratio
(Pooled) 93.164 106.902 6.769 74.693 6.769 122.215
Gender F F F F F M
Class Data
The most frequent genotype for Alu polymorphism TPA25, 51a & 182 was ID. Whilst
the most frequent for ACE was DD. For all STR's, allele D was most frequent. The
STR whose expected heterozygosity matched closest with observed heterozygosity
was TPA25. For ACE & 51a, the expected heterozygosity was higher than the
observed, whereas for 182 the observed was higher than the expected.
From the χ2 value we can conclude that with 95% confidence, all STRs except ACE
exist on HWE.
The combined match probability is 0.021, giving a probability of discrimination value
0.979. This database can thus be said to be 97.9% discriminatory.
The combined match probability for the class population is 0.021, indicating that
there is a 2.1% chance of two randomly selected individuals sharing the same
genotype for all four polymorphisms. The discrimination power is 0.979 concluding
Pooled Data
For every STR, the highest genotype frequency was ID and the most common allele
was D.
Expected heterozygosity was higher than observed heterozygosity for all STRs
however in each case, it was only by a small amount.
All STRs were found to be in HWE as all χ2 values were below the critical value (95%
confidence).
The combined match probability was calculated to be 0.017 giving rise to a combined
probability discrimination value of 0.983. Therefore this database can be said to be
98.3% discriminatory.
Pooled data shows similar results with a combined match probability of 0.017 and a
discrimination power of 0.983, giving a potential power of 98.3% to discriminate
between two random individuals. These high discriminatory values conclude that the
calculations are reliable.
Discussion
Calculations of allele frequencies showed that ACE was the only polymorphism
which did not exist in HWE. For a population to exist in HWE, several assumptions
should be met. These include random mating, no mutation or selection, no migration
and infinitely large population (Goodwin, Linacre, Hadi, 2007).
Our class population violates many of these assumptions. Firstly, it is not a randomly
selected population. Secondly, humans also violate the assumption of random
mating. Our class was also comprised of many individuals who had migrated to this
area. As a result of this, we would not expect our χ2 values to be in HWE. Looking at
our class data however, ACE is the only polymorphism which is not in HWE. This
can be explained due to the fact that our population was so small (n=67) that it was
not a good representation of the proportions of subgroups that it was comprised of.
Research has found that there are differences in allele frequencies amongst different
ethnicities (Ishigami, 1995). Our class population is comprised of different ethnicities.
This explains why, for each polymorphism, the difference between observed and
expected heterozygosity varies.
From the pooled results the expected heterozygosity values for the all the
polymorphisms are higher than the observed heterozygosity indicating that these
STRs may be useful as genetic markers in forensics.
From our own samples, sample 38 had the highest profile (0.247) frequency giving a
likelihood ratio of 4 to 1. However this only accounts for two polymorphisms, ACE
and TPA 25, as results for 51a and 182 were inconclusive. If we were to exclude this
sample and look only at fully conclusive samples, sample 39, with a profile frequency
of 0.026 was the most common with a likelihood ratio of 39 to 1. Sample 36 has the
lowest profile frequency 0.003 and a likelihood ratio of 289 to 1. This concludes that
within our class a higher percentage of people will have a similar profile frequency as
sample 39. Within the pooled population, excluding the sample with inconclusive
results, sample 39 has the highest profile frequency (0.013) and therefore a
likelihood ratio of 75 to 1. Sample 37 had the lowest profile frequency (0.009) and a
likelihood ratio of 107 to 1. Three out of four of our known samples had higher
likelihood ratio in the class population compared to pooled population. This could be
the result of analyzing a very small and diverse population. When analyzing the
marker for gender, all our known samples were correctly identified as being female.
This provides us with confidence that the process of determining the gender of
unknown samples was reliable. However the accuracy of gender determination can
be improved by analyzing the markers AluSTXa with AluSTYa. (Mastana, 2007).
Some of the samples did not produce eligible results on the gel electrophoresis for
particular markers. This may have been due to contamination of samples, errors
made while preparing samples or errors made during the preparation of materials for
electrophoresis.
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Appendices
a. You have been provided with 4 x 3mm diameter punches of the bloodstained
filter paper (your unknown sample)
b. Pipette 1ml of RNAse/DNAse free water into the tube.
c. Incubate at room temperature for 30 minutes.
d. Vortex mix for 5 seconds.
e. Centrifuge in a microcentrifuge at 13 000rpm for 2 minutes.
f. Without disturbing the pellet, carefully remove the supernatant, leaving
enough behind (20-30µl) to cover the pellet without disturbing it. If the
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a. Use 20% Chelex (w/v) prepared previously. Keep the solutions homogenous
using a magnetic stirrer.
b. Remove and discard the brush from the tube.
c. Vortex the sample for 5 seconds.
d. Centrifuge the sample for 2 mins @ maximum speed. Discard the
supernatant, leaving approx. 20-30µl residual fluid.
e. Add 170ul of 20% Chelex (w/v) to give a final volume of 200µl and incubate
30 mins @ 56°C (water bath).
f. Vortex (10 secs) and place in Dry Block (~ 100 °C) for 8 mins.
g. Vortex again and centrifuge for 3 mins @ max speed (13000 RPM).
h. Label a fresh microfuge tube with your ID number.
i. Remove supernatant to a clean labelled 1.5ml microfuge tube.
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Primers
Add 100ml, 1X ethidium bromide -TBE pH8.3 and cover with cling film.
Pierce the cling film and swirl the mixture before placing in a microwave for 1minute.
Remove and swirl again. If the solution has not gone clear then heat for a further 1
minute, checking that the solution has not boiled over.
Allow the agarose to cool to hand hot and gently pour into the mould, with the combs
in place and allow to set (Approximately 30 minutes).
References
Batzer, M.A., Arcot, S.S., Phinney, J.W., Alegria-Hartman, M., Kass, D.H.,
Milligan,S.M., Kimpton, C., Gill, P., Hochmeister, M., Ioannou, P.A., Herrera, R.J.,
Boudreau, D.A., Scheer, W.D., Keats, B.J., Deininger, P.L., Stoneking, M., 1996.
Genetic variation of recent Alu insertions in human populations. Journal of Molecular
Evolution, 42, pp. 22-29.
Batzer, M.A., Stoneking, M., Alegria-Hartman, M., Bazan, H., Kass, D.H., Shaikh,
T.H., Novick, G.E., Ioannou, P.A., Scheer, W.D., Herrera, R.J., 1994. African origin
of human-specific polymorphic Alu insertions. Proceedings of the National Academy
of Sciences of the United States of America, 91(25), pp.12288-12292.
Feng, Y., Niu, T., Chen, C., Li, Q., Qian, R., Wang, G., Xu, X.,2002. Insertion/Deletion
Polymorphism of the ACE Gene Is Associated With Type 2 Diabetes. Diabetes, 51,
pp.1986-1988.
Gasper, P., Seixas, S., Rocha, J., 2004. Genetic Variation in a Compound Short
Tandem Repeat/Alu Haplotype System at the SB19.3 Locus: Properties and
Interpretation. Human Biology, 76(2), pp.277-287.
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Roy, A.M., Carroll, M.L., Kass, D.H., Nguyen, S.V., Salem, A.H., Batzer, M.A.,
Deininger, P.L., 1999. Recently integrated human Alu repeats: finding needles in the
haystack. Genetica, 107(1-3), pp.149-161.
Watkins, W.S., Rogers, A.R., Ostler, C.T., Wooding, S., Bamshad, M.J., et al., 2003.
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polymorphisms. Genome, 13, pp.1607-1618.
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