DNA profiling practical

Question 1

What is DNA profiling

Answer 1

DNA profiling is the use of molecular genetic methods to determine the exact
genotype of a DNA sample to distinguish one human from another

Question 2

DNA sequences

Answer 2

The DNA sequences used in forensic DNA profiling are non-coding regions that contain segments of short tandem repeats or STRs.

Question 3

STRs

Answer 3

STRs are very short DNA sequences that are repeated in direct head-to-tail fashion

Question 4

How are STR alleles detected?

Answer 4

The key to DNA profiling is amplification of the copies present in the small amounts of evidentiary DNA by polymerase chain
reaction (PCR

Question 5

What is the power of discrimination?

Answer 5

In real crime scene applications, DNA profiling is performed at a number of different loci to improve the power of discrimination of the testing. In simple terms, the power of discrimination is the ability of the typing to discriminate between different individuals

Question 6

Step one

Answer 6

Label five PCR tubes CS, A, B, C, or D, and include your group name or initials as well. Place each PCR tube on ice.

Question 7

Step two

Answer 7

Transfer 20 μl of the appropriate template DNA into the correctly labelled tube. Important: use a
fresh pipette tip for each DNA sample.
E.g. Suspect A DNA in tube A

Question 8

Step three

Answer 8

Transfer 20 μl of the blue MMP (master mix +primers) into each of these 5 PCR tubes already containing the template DNA. Pipette up and down to mix. Cap each tube after adding blue MMP. Important: use a fresh pipet tip each time. Immediately cap each tube after adding MMP.

Question 9

Step four

Answer 9

When instructed to do so, place your tubes in the thermal cycler. Your instructor will program the thermal cycler for PCR

Question 10

Step five

Answer 10

The PCR instrument will amplify your crime scene samples so that they can be analyzed using gel electrophoresis. Mark your samples so that you will recognize them, as you will need the in the following week.

Question 11

Step six

Answer 11

To analyze the PCR products you will need to use 1X TBE buffer and 3% agarose gels in 1X TBE buffer for suitable resolution of the short PCR products.

Question 12

Step seven

Answer 12

Immerse the gel to the gel tank containing 1X TBE buffer

Question 13

Step eight

Answer 13

Add 12μl of size marker (CSI Allelic ladder) to lane 1 (first well).

Question 14

Step nine

Answer 14

Pipette at least 12μl of each PCR product to individual wells (2nd, 3rd, 4th etc.). Do not pipette on the same well where you already have the size marker or another sample. Do not overload the wells or load multiple samples in the same well.

Question 15

Step ten

Answer 15

Run the gel with constant voltage of 10V/cm of gel until the blue marker dye has advanced at least half way the gel. Thus, for example if your gel is 15 cm gel use 150V constant voltage.

Question 16

Step eleven

Answer 16

Visualize the gel on Gel Workstation and save the image on USB stick.