DNA In Your Bones !

Welcome back ! 

Today, we shall look into DNA profiling ! DNA is in your blood and in your bones too! It's fascinating to know that DNA makes you who you are today and helps a great deal in forensic science. 

Picture adapted from :http://dnaphenomena.blogspot.com/2011/05/dna-profiling.html 

In the past, the analysis of DNA has been successfully employed in criminal cases, disaster victim identification and paternity testing. DNA profiling reveals a suite of variations in the genetic code that, taken together, constitute an individual’s unique DNA profile. In cases of mass disaster, the remains are usually skeletonised where the bodies have been badly degraded and the bodily tissues have decayed. DNA profile has to be performed from mitochondrial DNA (mtDNA) extraction because nuclear DNA can hardly be extracted from these remains.

The sequence of mtDNA is entirely functional and highly conserved, so there is very little variation between individuals. The focus in forensic analysis of mtDNA is on the hypervariable regions – HV1 and HV2 – within the control region (a 1000 base pair long non-coding D-loop). The variations within these regions are generally single nucleotide polymorphisms. mtDNA is also subjected to high rate of mutation due to its lack of DNA reparation, causing variation between individuals.

Step 1 – DNA extraction

1.   Lyse sample cells in a buffer solution.

2.   Centrifuge the denatured proteins and fats.

3.   Pass the cleared lysate through a column containing a positively charged medium that   binds to the DNA.

4.   Remove contaminating proteins, fats and salts through several washes.

5.   Recover the DNA in a buffer solution.

Step 2 – PCR amplification of HV1 and HV2

Picture adapted from : http://www.medpreponline.com/2008/07/polymerase-chain-reaction-pcr-paternity.html

The minute mtDNA obtained has to be amplified by Polymerase Chain Reaction (PCR). PCR enables a single copy of a DNA fragment to be amplified to millions of copies in just a few hours.

Component required	Function
Oligonucleotide primer x2	Mark complementary DNA target to be amplified. ―     Base sequence of one primer binds to one side of the target, the other primer binds to the other side of the target, with the DNA between the primers.
Fluorescent tags	Visualize amplified DNA in electrophoresis.
Polymerase enzyme	Allows DNA strand to be copied by adding nucleotides to 3’ end of primers.
Reaction buffer with MgCl	Ensures ideal conditions for functioning of polymerase enzyme.
Deoxyribonucleotides	Builds DNA molecule.
Template DNA	Constructs messenger RNA.

The PCR process is conducted in a small, plastic centrifuge tube, and the cycle consists of:

1.       Denaturation: heat the sample to 94-95°C for 30 seconds.

―     Allows primers to access by breaking hydrogen bonds which separates the double-stranded DNA.

2.       Annealing: keep the sample at 50-65°C.

―     Allows hydrogen bonds to form between the primers and the complementary DNA sequence.

3.       Extension: heat the sample to 72°C for a duration depending on the length of the DNA strand to be amplified and the speed of the polymerase enzyme which builds up the strand, and then add deoxynucleotide triphosphates to the 3’ end of the primer.

―     Elongation stage.

Each PCR cycle takes about 5 minutes, with the amount of the original sequence being doubled. The process can then be repeated as necessary to obtain the desire amount of sample.

Step 3 – DNA sequencing

Sanger sequencing establishes the base pair sequence of the HV1 and HV2 regions. The steps involved are:

1.       Initiate DNA synthesis using a labeled primer.

2.       Add four dideoxy nucleotides and randomly arrest synthesis.

3.       Separate fragments produced using electrophoresis.

4.       Convert band patterns into DNA sequence using specialized software.

5.       Compare results with the Cambridge Reference Sequence to establish potential similarities and differences.

mtDNA enables the analysis of even highly degraded samples. If a victim is severely decomposed to the point that it is not possible to successfully extract a DNA profile using nuclear DNA, mtDNA is generally used. Moreover, it requires only a very small amount of sample.

Reference : http://forensicsciencecentral.co.uk/dna.shtml

Here's a short video clip on DNA profiling :) We hope you'll be able to comprehend what we have discussed with you so far. Enjoy the video !