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Analysis of Variation in the Human Genome using mtDNA,

amelogenin, Transposable element, VNTR, STR and RFLP


Primers
Muhammad Huzaifah bin Abd Rashid, Department of Biochemistry, Imperial College London, South
Kensington campus.

Here, we have a sample of DNA taken from a suspect extracted from a tissue found underneath
the fingernails in one of a murder victim and a set of DNA samples taken from the whole class.
Using some of the indispensable techniques in investigating the DNA samples such as Polymerase
Chain Reaction, Southern Blotting, DNA sequencing and analyzing variations at several different
loci, we can construct a genetic map to study the relationship between different populations or
individuals.

DNA Amplification and Gel Analysis Identities: 464/559 nucleotides match which
Using polymerase chain reaction (PCR), all the make it 84% similarities.
samples, including the suspect DNA are
amplified using different primers. The PCR Sample 3
products are then analyzed using gel
electrophoresis. Here are the results and Identities: 637/662 nucleotides match which
some discussions regarding the experiment make it 97% similarities.
using different type of primers.
Haplogroups analysis
1) Mitochondrial DNA (mtDNA)
From sample 1, from all the polymorphisms
Comparing three DNA sequences with the found, one substitution was found in position
suspect DNA: 16270 where C was substituted with T.

Sample 1 From sample 3, we found polymorphisms that


occur at position 16126 and 16294.
Identities: 639/662 nucleotides match which
make it 97% similarities. Unfortunately, for sample 2, the sequencing
quality was so bad; hence, there are too many
After analyzing the sequence, we can be sure unreliable sequencing data. Hence, we cannot
that there are real polymorphisms at certain determine confidently the haplogroup.
nucleotides especially with number 156, 203,
264. However, the difference in the ends of From the suspect DNA, we don’t have enough
the sequence may be due to poor quality such information to deduce the haplogroup.
as at nucleotides number 32, 635 and
onwards. Samples Haplogroup
1 UK
It clearly be seen that there are 2 Cannot be determined
polymorphisms in this sample compared to 3 T
the suspect. They are clearly not the same Suspect Cannot be determined
person.
2) Amelogenin
Sample 2
From the gel result, it is clearly seen two
bands from sample 1 and only one band from
sample 2 and 3. The suspect DNA forms two
bands on the gel electrophoresis.

The band with size around 433 bp amplified


from AMELX while the band with size 253 bp
is the product of AMELY amplification.
From the gel above, from sample 1, we can
As from the gel, we can conclude that sample clearly see a band with size about 424 bp. In
1 is a male while samples 2 and 3 are female. samples 2, 3 and the suspect, there is a band
The suspect is also a male, so, this rule out seen in the region of size about 113 bp.
both samples 2 and 3 from the list of suspects.
Hence, from the observations, we can deduce
From the gel, it can be seen that in sample 1, that individual 1 is homozygous for the
the larger band correspond to AMELX is insertion (+/+). Conversely, samples 2, 3 and
slightly faint compared to the other band. This the suspect are homozygous lacking the
might due to changes to the primer binding insertion (-/-).
site, hence, less primer binds and less
amplification occurs within that region. However, the result may be not quite reliable.
Larger fragments may difficult to amplify as
Amelogenin test is a good start in determining the Taq Polymerase has a low processivity.
the identity of the suspect. However, This means that this enzyme detach quickly
sometimes, the test can be quite misleading. before completing the elongation reaction.
For an example, if there are mutations in Y- Hence, there may be some larger fragments
derived fragments, this may result in failure of of other samples that fail to amplify to be
amplification of AMELY making the gender seen on the gel.
determination misleading.
Genotype Frequencies (class results):
In a scenario where the male and female Genotype Frequency
samples are mixed, we can mix the mixture +/+ 5/33
with cell lysis buffer, proteinase K, SDS and +/- 5/33
EDTA, then, centrifuge the mixture to -/- 23/33
separate the female sample as the
supernatant and the male fragment in the Allele Frequencies
pellet. Allele Frequency
+ 15/66
3) TPA25 (Transposable Element) - 51/66

Genotype Frequencies (expected)


Genotype Frequency
+/+ 2/44
+/- 12/44 Hence, it can be concluded that individual 1 is
-/- 30/44 heterozygous, while individual 3 is
homozygous for 21 repeats and the suspect is
The χ2 value for this set of data is 10.67013. homozygous for 24 repeats.
From this value, it can be concluded that the
genotype frequency of this population is not From the samples of the University of Utah,
consistent with Hardy-Weinberg Equilibrium. individual with the same D1S80 pattern as our
suspect are individual 6 and 10. However,
When the class results genotype frequency is there are possibilities that individual 21, 22,
compared with Asian genotype frequency, the 23 also share the same repeats due to slight
χ2 value calculated is 6.55316 which mean difference that may arise from the techniques
that the genotype distributions in both of the used. Other than these, all other samples can
populations are significantly different. be safely excluded.

4) D1S80 (Variable Number of Tandem The allele frequency of 24 repeats is 0.335 in


Repeats – VNTR) the UK. Hence, the probability of finding a
homozygous individual with 24 repeats is
(0.335)2 which is 0.112. The probability of
finding individual with the criteria is 0.0484 in
China.

5) Short Tandem Repeat – STR

20
The frequency of finding allele 164:
72

16
The frequency of finding allele 168:
72

20 16 5
Probability of finding a match: × =
72 72 81
From the above result, for each sample, we
can approximate the size of bands seen. The = 6.17 %
approximate sizes are recorded in this table:
6) DRD2 (Restriction Fragment Length
Sample Size of bands Number of Polymorphism – RFLP)
Repeats
1 550, 650 24, 31 Sample 1
2 No band -
3 500 21
Suspect 550 24

We can also see some faint bands of size


about 330 bp in (sample 1) and about 110 bp
(suspect). However, we do not consider these
as our bands of interest because their sizes
are not within the range that we expected
from the amplification. These bands might
occur due to non-specific amplifications.
There was no band seen from sample 2.
Sample 2 might have failed to amplify.
This shows that sample 2 are heterozygous for
these alleles. From the double digest, we can
clearly observe two smaller bands and one
Sample 2 band correspond to larger fragment. This
indicates that both restriction sites are on the
same chromosome.

In summary, we deduced these conditions:

Sample Genotype
1 T+B+/T+B+
2 T+B+/T-B-
3 T+B+/T+B+
Suspect T+B+/T+B+

The haplotype and allele frequencies from the


class this year is shown below:
Sample 3
Haplotyp Frequency Allele Frequency
e
+ +
TB 38/58 T+ 40/116
T-B- 17/58 T- 18/116
T+B- 2/58 B+ 39/116
T-B+ 1/58 B- 19/116

Linkage disequilibrium of SNPs:

38/58 – (40/116 x 39/116) = 0.539


= 53.9 % LD
Suspect DNA

From the gel from sample 1, we observed that


the entire samples are digested both in Taq1
and Bcl1, indicating that sample 1 is
homozygous for both of these sites. The same
observation can also be seen in sample 3 and
the suspect.

However, the digest from sample 2 is


different. It can clearly be seen 2 small bands
on the gel for both samples digested with
Taq1 and Bcl1. However, there are also a band
correspond to larger fragment on both digest.

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