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VNTR Human DNA

Using PCR

CHE 447-01
By Grant Akalonu
Grant Akalonu VNTR Human DNA Typing Using PCR

Objective/Introduction

In forensics, DNA fingerprinting is a laboratory technique used to relate biological

evidence from a crime scene to suspects and victims, in a qualitative manner. DNA fingerprints

for specific individuals are determined by analyzing regions of chromosomes that vary greatly

among individuals These varying regions in chromosomal sequences are known as DNA

polymorphs. Analysis of polymorphic regions of DNA are also used to determine family

relationships such as maternity, paternity, or to identify human remains.

DNA fingerprinting first requires collection of hair, or bodily fluids from a crime scene

or victim. The sample is then stained and treated with a detergent to rupture the cell membranes

to acquire the DNA. The polymerase chain reaction (PCR) is utilized to amplify and examine

DNA regions of high polymorphic character. Polymorphic DNA regions fall into two categories,

VNTR (Variable Number of Tandem Repeats), and STR (Short Tandem Repeats). In this

experiment, the VNTR of interest is D1S80, which exists on chromosome 1 and has a 16

nucleotide sequence that is repeated anywhere between 16 and 40 times. After the DNA was

amplified at the D1S80 locus by PCR, it was visualized on agarose gel for analysis. The number

of bands on the the agarose gel indicates whether the student is heterozygous (two bands) or

homozygous (one band) for the D1S80 genotype.

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Grant Akalonu VNTR Human DNA Typing Using PCR

Materials & Methods

Part 1:VNTR Human DNA Typing using PCR

First, students rinsed their mouths vigorously for 60 seconds with 10 mL of saline water.

Then, the rinsed saline solution was swirled in a cup to suspend the cells and transferred to a a

centrifuge tube. The solution was then centrifuged at 800 rpm for two minutes. This was

repeated a number of times to create a cell pellet large enough for the next part of the procedure.

Then, the supernatant liquid above the pellet was removed and 140 L of lyse buffer was added

to the pellet. The mixture was then vortexed and incubated in a water bath at 55C for 15

minutes. The mixture was vortexed again and then incubated at 99C for 15 minutes. Next the

solution was centrifuged again for 2 minutes at 6000 rpm. 80 L of the supernatant was collected

and store on ice. Then, 20 L of primer, 5 L of the extracted DNA and a PCR Edvobead was

added to a 0.2 mL PCR tube. A similar PCR tube was prepared for the control DNA. and

another students DNA. This solution was mixed to ensure the dissolution of the bead, and then

quick centrifuged. The DNA samples were then amplified using the PCR, and its settings are

summarized in Table 1 and repeated for 35 cycles.

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Grant Akalonu VNTR Human DNA Typing Using PCR

PCR Step Temperature (C) Duration (s)

Initial Denaturation 94 240

Denaturation 94 30

Annealing 65 30

Extension 72 30

Final Extension 72 240

Table 1: PCR Settings Summary

Part 2: Agarose Gel for Human DNA Typing Using PCR

After the PCR is complete, 5L of 10x gel loading solution was added to the sample. The

gel was prepared by another group of students at the beginning of the lab period. 50x TAE

electrophoresis buffer was diluted to 1x, and 1.5% agarose gel was prepared by mixing 0.38g of

UltraSpec-Agarose powder in 25 Ml 1x TAE electrophoresis buffer. 30 L of each sample was

loaded into the gel after it solidified and ran at 140V for 45 minutes. The gel was then stained

with ethidium bromide and then viewed for analysis.

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Grant Akalonu VNTR Human DNA Typing Using PCR

Results

PCR Agarose Gel Result

Figure 1: PCR Agarose Gel Result.

Lane Number of Base Pairs in the Sample

1 (DNA Ladder) 200

2 (Control) 200

3 Grant Akalonu DNA sample 200

4 Damien DNA sample 200

Table 2: Estimation of Number of Base Pairs from Each Sample.

Fig.1. PCR Gel Result of VNTR Typing

Discussion & Conclusion

The human fingerprinting method described in this experiment was expected to show

differences in the genotypes of students by showing whether they were heterozygous or

homozygous to the DS180 genotype. However, a malfunction with the PCR machine did not

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Grant Akalonu VNTR Human DNA Typing Using PCR

allow for the visualization of different bands among students, as all of the gels in the class had

the same bands for every student, representing 200 base pairs.

Answers to Required Questions*

1) There are two bands on the agarose gel from my DNA sample, which indicate that I

am heterozygous for the D1S80 genotype. It appears that George is also heterozygous

for the same genotype, as indicated by his bands at 200 and 600 base pairs. This

similarity in our results could possibly indicate a common ancestry between us, or

could simple be coincidental.

2) Polymorphic DNA refers to regions of chromosomes that vary greatly among

individuals. The main characteristic of these chromosomal regions that vary among

individuals is their length. The amount of base pairs that make up polymorphic DNA

in one individual is highly unlikely to be observed in another individual. Therefore,

polymorphic regions of DNA can be used to match samples of DNA from various

sources to an individual.

3) CODIS is the acronym for the Combined DNA Index System. It is a computer
system containing DNA fingerprints from crime scenes. It enables federal, state, and
local forensic laboratories to exchange and compare DNA profiles electronically,
thereby linking serial violent crimes to each other and to known offenders.
4) An short tandem repeat (STR) is a DNA sequence that varies from person to person

and is 2-4 base pairs in length. A variable number tandem repeat (VNTR) is a DNA

sequence varying from person to person but ranges from 15-70 base pairs in length.

Short tandem repeats are preferred in law enforcement, because they require a smaller

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Grant Akalonu VNTR Human DNA Typing Using PCR

amount of template DNA. This allows law enforcement to obtain viable experimental

results from samples that have degraded.