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

Sample 1 From sample 3, we found polymorphisms that

Identities: 639/662 nucleotides match which
make it 97% similarities.
After analyzing the sequence, we can be sure
that there are real polymorphisms at certain
nucleotides especially with number 156, 203,
264. However, the difference in the ends of
the sequence may be due to poor quality such
as at nucleotides number 32, 635 and
onwards.
It clearly be seen that there are
polymorphisms in this sample compared to
the suspect. They are clearly not the same
person.
Sample 2
occur at position 16126 and 16294.
Unfortunately, for sample 2, the sequencing
quality was so bad; hence, there are too many
unreliable sequencing data. Hence, we cannot
determine confidently the haplogroup.
From the suspect DNA, we don’t have enough
information to deduce the haplogroup.
Samples Haplogroup
1 UK
2 Cannot be determined
3 T
Suspect Cannot be determined
2) Amelogenin
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.
As from the gel, we can conclude that sample
1 is a male while samples 2 and 3 are female.
The suspect is also a male, so, this rule out
both samples 2 and 3 from the list of suspects.
From the gel, it can be seen that in sample 1,
the larger band correspond to AMELX is
slightly faint compared to the other band. This
might due to changes to the primer binding
site, hence, less primer binds and less
amplification occurs within that region.
Amelogenin test is a good start in determining
the identity of the suspect. However,
sometimes, the test can be quite misleading.
For an example, if there are mutations in Y-
derived fragments, this may result in failure of
amplification of AMELY making the gender
determination misleading.
In a scenario where the male and female
samples are mixed, we can mix the mixture
with cell lysis buffer, proteinase K, SDS and
EDTA, then, centrifuge the mixture to
separate the female sample as the
supernatant and the male fragment in the
pellet.
3) TPA25 (Transposable Element)
From the gel above, from sample 1, we can
clearly see a band with size about 424 bp. In
samples 2, 3 and the suspect, there is a band
seen in the region of size about 113 bp.
Hence, from the observations, we can deduce
that individual 1 is homozygous for the
insertion (+/+). Conversely, samples 2, 3 and
the suspect are homozygous lacking the
insertion (-/-).
However, the result may be not quite reliable.
Larger fragments may difficult to amplify as
the Taq Polymerase has a low processivity.
This means that this enzyme detach quickly
before completing the elongation reaction.
Hence, there may be some larger fragments
of other samples that fail to amplify to be
seen on the gel.
Genotype Frequencies (class results):
Genotype Frequency
+/+ 5/33
+/- 5/33
-/- 23/33
Allele Frequencies
Allele Frequency
+ 15/66
- 51/66
Genotype Frequencies (expected)
Genotype Frequency
+/+ 2/44
 +/- 12/44
-/- 30/44
The χ2 value for this set of data is 10.67013.
From this value, it can be concluded that the
genotype frequency of this population is not
consistent with Hardy-Weinberg Equilibrium.
When the class results genotype frequency is
compared with Asian genotype frequency, the
χ2 value calculated is 6.55316 which mean
that the genotype distributions in both of the
populations are significantly different.
4) D1S80 (Variable Number of Tandem
Repeats – VNTR)
From the above result, for each sample, we
can approximate the size of bands seen. The
approximate sizes are recorded in this table:
Sample Size of bands Number of
Repeats
1 550, 650 24, 31
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.
Hence, it can be concluded that individual 1 is
heterozygous, while individual 3 is
homozygous for 21 repeats and the suspect is
homozygous for 24 repeats.
From the samples of the University of Utah,
individual with the same D1S80 pattern as our
suspect are individual 6 and 10. However,
there are possibilities that individual 21, 22,
23 also share the same repeats due to slight
difference that may arise from the techniques
used. Other than these, all other samples can
be safely excluded.
The allele frequency of 24 repeats is 0.335 in
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
= 6.17 %
6) DRD2 (Restriction Fragment Length
Polymorphism – RFLP)
Sample 1
 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.