Analysis of Microsatellite Loci on Chromosome 15
Melissa Bochnowicz
Partner: Elizabeth Davidson
Instructor: Dr. Carlini & Ben Gamache
Due: Monday, April 29, 2013
Biology-356 Section 001H
Abstract:
Genomic DNA was extracted, then analyzed through PCR amplification and gel
electrophoresis for 3 microsatellite loci on chromosome 15. These samples were then
measured and compiled according to genotype in order to determine both allelic
frequencies and whether the sample studied (the class) was in Hardy-Weinberg
equilibrium at one of the loci. Based on the data, 17 of 28 genotypes were observed with
7 different alleles at the D15S657 locus, 28 of 66 were observed with 11 different alleles
at the D15S652 locus, and 16 of 28 were observed with 7 different alleles at the D15S655
locus; of a possible 51,744 possible genotypes, only 43 were present. Allelic frequency
was calculated by dividing the number of times an allele occurred by the total number of
alleles present (in this case, 86 alleles were present). After completing a chi-squared test
of equilibrium at the D15S657 locus in which chi-squared equaled 12.63460511, p
equaled 0.9914 and there was 27 degrees of freedom, it was determined that the sample
was indeed in Hardy-Weinberg equilibrium.
Introduction:
Microsatellites are a category of simple sequence repeats within the DNA that
account for roughly 3% of the human genome. Most of these loci are not transcribed into
RNA and are considered highly polymorphic since the mutations that accumulate within
this portion of the DNA does not go on to affect the phenotype and therefore are not
repaired as often as other, coding portions of the DNA. This level of polymorphism is
due to the highly variable amount of copies per repeat unit in any one allele; these are
also referred to as Variable Number Tandem Repeats (VNTR) and arise due to a defect in
DNA polymerase that results in an increased number of repeats. Since DNA polymerase
does not fix these mutations, they often times result in the lengthening and shortening of
microsatellites, which in turn increases the number of repeating units (Lai & Sun 2003).
Thus, each locus alone can contain hundreds of different alleles, making most individuals
heterozygous at these points in the DNA and giving them all a unique DNA profile
(fingerprint) if enough loci are analyzed. Since each individual (other than identical
twins) has a unique DNA fingerprint, microsatellite analysis is typically used in criminal
forensics and paternity suits to identify the proper individuals (Carlini 2013).
For this experiment, student’s genomic DNA was extracted then analyzed through
PCR amplification and gel electrophoresis in order to determine whether the class was in
Hardy-Weinberg equilibrium. This was done using three distinct primer pairs per loci
when completing the PCR amplification, analyzing the genotypic data gathered for the
entire class and comparing this to the expected results of Hardy-Weinberg equilibrium. It
was hypothesized that the class would not in Hardy-Weinberg equilibrium due to the size
and nature of the sample studied.
Materials & Methods:
During Week 1, a 0.5X dilution of genomic DNA (obtained earlier in the
semester) was created by transferring 5 μl of the DNA to a tube containing 5 μl of
ddH2O. A 0.25X dilution was prepared by transferring 5 μl of the 0.5X diluted solution
from the previous step to a different tube containing 5 μl of ddH2O. Both tubes were
gently mixed before having 1 μl from each dilution pipetted into separate tubes. Once
complete, the samples were placed in a thermocycler to undergo the thermal profile
shown below. The samples were then kept at 4°C for the next 2 weeks.
Thermal Profile
Step
Temperature
Time
Denaturation
95°C
5:00
5 cycles decreasing anneal
95°C
0:45
temp. 2°C each cycle:
68°C
5:00
72°C
1:00
5 cycles decreasing anneal
95°C
0:45
temp. 2°C each cycle:
58°C
2:00
72°C
1:00
95°C
0:45
50°C
2:00
72°C
1:00
72°C
5:00
25 cycles
Final Polymerization
After 2 weeks, the diluted genomic DNA was loaded into a gel and run for
approximately 45 minutes. Images of the gel were then uploaded and the DNA profile
analyzed accordingly. First, the straight-line vertical distance was measured for each of
the 7 bands for both the far left ladder and the far right ladder. These values were then
compiled and a linear regression performed in order to determine the appropriate
equation for calculating the size of the DNA bands. After measuring the distance from
the top of the gel for each of the 10 subsequent samples, the size of each allele was
calculated using the above-determined equations (depending which ladder the sample
was closer to determined which equation was used). Once complete, all data was
compiled and compared.
Next, a series of questions were answered in order to first determine allelic
frequency, then whether the genotypes observed within the class were in HardyWeinberg equilibrium. To start, the number of different alleles was identified, then the
number of possible different genotypes calculated. This value was compared to the actual
number of genotypes observed for the class. Then allelic frequency was calculated by
counting the number of times a single allele appeared and dividing this value by the total
number of alleles present (in this case, 86 alleles were present). Finally, the expected
frequencies for Hardy-Weinberg equilibrium were calculated for the 15S657 locus only
using the expanded form of the below equation:
( p + q + r + s + t + u + v)2 = 1
where each variable represents the genotypic frequencies of each of the 7
homozygous genotypes, and the multi-variable portions refer to the frequencies of the
heterozygotes. These values were multiplied by the sample size in order to accurately
determine the expected number of students to have a single genotype. A chi-squared test
was then performed to determine whether the observed data was in Hardy-Weinberg
equilibrium.
Results:
Table 1: Allele Frequency for Chromosome 15 at 3 Loci
D15S657 Locus
D15S652 Locus
D15S655 Locus
Allele
Frequency Allele
Frequency Allele
Frequency
344
0.058
300
0.035
251
0.012
348
0.279
303
0.221
254
0.209
352
0.198
306
0.047
257
0.302
356
0.233
309
0.081
260
0.128
360
0.116
312
0.093
263
0.035
364
0.105
315
0.116
266
0.140
368
0.012
318
0.058
269
0.174
321
324
327
330
0.198
0.093
0.023
0.035
Table 2: Test for Hardy-Weinberg Equilibrium at D15S657 Locus
Observed
Expected
Number
Expected
Number of of
Genotype Frequency Students
Students
(O-E)2/E
344/344
344/348
344/352
344/356
344/360
344/364
344/368
348/348
348/352
348/356
348/360
348/364
348/368
352/352
352/356
352/360
352/364
352/368
356/356
356/360
356/364
356/368
360/360
360/364
360/368
364/364
364/368
368/368
0.0038
0.03245
0.02299
0.027028
0.013456
0.01218
0.001392
0.077841
0.110484
0.130014
0.064728
0.05859
0.006696
0.039204
0.092268
0.045936
0.04158
0.004752
0.054289
0.054056
0.04893
0.005592
0.013456
0.02436
0.002784
0.011025
0.00252
0.000144
0.1453
1.3953
0.9884
1.162204
0.578608
0.52374
0.059856
3.347163
4.750812
5.590602
2.783304
2.51937
0.287928
1.685772
3.967524
1.975248
1.78794
0.204336
2.334427
2.324408
2.10399
0.240456
0.578608
1.04748
0.119712
0.474075
0.10836
0.006192
0
0.1453
3
1.8453
1
0.0001
1
0.0226381407
0
0.578608
0
0.52374
0
0.059856
4
0.1273305628
4
0.1186573283
4
0.4525478155
2
0.220448944
2
0.1070685119
1
1.761018495
2
0.0585721177
5
0.2686831108
2 0.0003101694086
1
0.3472428849
0
0.204336
2
0.0479095805
3
0.1963616329
3
0.3815768707
0
0.240456
0
0.578608
3
3.63952949
0
0.119712
0
0.474075
0
0.10836
0
0.006192
χ2
Degrees
12.63460511
27
of
Freedom
P-Value
0.9914
Discussion:
A comparison of the alleles at each locus showed that at the D15S657 locus, the
348 allele occurred most frequently, appearing 24 times within the sample resulting in a
frequency of 0.279; at the D15S652 locus, the 303 allele occurred most frequently,
appearing 19 times within the sample resulting in a frequency of 0.221; and at the
D15S655 locus, the 357 allele occurred most frequently, appearing 26 times within the
sample resulting in a frequency of 0.302.
For the D15S657 locus, 7 alleles were present and of the 28 possible genotypes,
17 were observed. At the D15S652 locus, 11 alleles were present and of the 66 possible
genotypes, 28 were observed. Finally, at the D15S655 locus, 7 alleles were present and of
the 28 possible genotypes 16 were observed. Many students shared the same genotype
within, but there were only 2 pairs of student (TA13/TA8 & F7/TP13) that shared
genotypes at 2 loci, in this case the D15S657 locus and the D15S655 locus. No students
shared the same genotype at all 3 loci.
After completing the chi-squared analysis, it was determined that the genotypic
frequencies at the D15S657 locus were indeed in accordance with Hardy-Weinberg
equilibrium, thus proving the hypothesis true. The chi-squared value of the completed test
was 12.63460511 with a p-value of 0.9914 at 27 degrees of freedom. This means that
there was a 99.14% chance that the observed and expected genotypic frequencies for the
sample were caused at random. There was no significant difference between observed
and expected, thus meaning the sample was in Hardy-Weinberg equilibrium. Any of the
deviations observed can be explained by the finite sample size (population), random
mutations or natural selection.
Using only these three microsatellite loci, it would be possible to correctly
identify any student based solely on an unknown DNA sample. This is possible since no
two students share the same genotype at all three of the loci observed. If only the
D15S657 and D15S655 loci were observed, a definitive identity would not be reached
because multiple students share the same genotype at these loci. Though observing only 3
loci works for this finite sample, a greater diversity of loci should be viewed when
searching for an individual in a larger population due to the greater possibility of similar
genotypes occurring at only 3 loci.
References:
Carlini D. 2013. PCR Amplification of Three Microsatellite Loci on Chromosome 15.
Lab Handout.
Carlini D. 2013. Analysis of Microsatellite Data. Lab Handout.
Lai Y, Sun F. 2003. The Relationship Between Microsatellite Slippage Mutation Rate
and the Number of Repeat Units. Molecular Biology and Evolution 20(12): 21232131.