The human population is so diverse- some people have black hair, some blonde, some auburn and some red- and yet we are so similar at the biological level, after all, we all do have two legs and two hands! You would be surprised to know that the diversity amongst humans is caused by only 1% of the DNA. 99% of human DNA is shared by all of mankind! How much does this 1% account for? Around 3 billion base pairs that make us different from one another- and unique.
We often relate humans to their characteristic traits, features and habits. However, these traits or habits can be found in others as well (even if it’s rare). So, what makes us, “us”? Our DNA fingerprint. It is what makes us unique.
DNA fingerprinting or DNA profiling is a method of identifying a specific individual. It has been used by forensics to solve crime cases, it is used for paternity tests and even for finding genetic links in hereditary illnesses.
In the summer of 1984, Alec Jeffreys- a genetic scientist of Leicester University- was working on family lineages to track hereditary diseases. He had stumbled upon a fragment of DNA that was repeated in every cell. These repeats are known as minisatelites or variable number tandem repeats (VNTRs). He wondered if he could count the number of repeats in each cell. For doing so, he devised an experiment.
Step 1- Extraction of DNA from sample.
The sample could be blood, saliva, hair etc. Usually detergents are used for this process that form micelles with the protein and lipid content of the cell membrane, thus breaking the cell membrane. This leaves the DNA content exposed to the environment.
Step 2- Cutting the DNA
Molecular scissors or restriction enzymes are used to cut DNA at specific sites. Restriction enzymes recognize a particular segment of the DNA and cut only at a specific site. For example, if a restriction enzyme cuts between C and G of a segment ATCG then the cut will look like this-
The result of this cleavage are fragments of varying lengths known as Restricted Fragment Length Polymorphisms (RFLP). A polymorphism literally means the existence of different forms of a particular entity. These RFLPs contain VNTRs.
Step 3- Separation on the basis of size
DNA is negatively charged due to the presence of phosphate group. When loaded into a gel based matrix it will travel towards the anode, once voltage is applied. The shortest DNA fragment will travel the furthest and the longest one, the least. The gel acts as a sieve here.
Step 4- Denaturing the fragments
The DNA fragments are denatured by transferring them to an alkaline medium. This breaks the DNA fragments into single strands. This step is done to perform the next step- southern blotting.
Step 5- Southern Blotting
A membrane (usually nitrocellulose) is placed on the gel and light pressure is applied on it by placing a stack of paper. The moisture is absorbed by the paper and the single strands are transferred to the membrane.
Step 6- Pre-hybridization
Pre- hybridization is a blocking step before addition of probe. It prevents the non-specific attachment of probe to the membrane.
Step 7- Hybridization with DNA probe
DNA probe is nothing but a single stranded DNA which is complementary to the desired DNA sequence. The probes are tagged with radioactive or chromogenic substance for easy detection.
Step 8- Autoradiography
After washing away the excess probes, the membrane is studied using autoradiography. The hybridization pattern developed on the X- ray film is known as the DNA fingerprint.
Let us investigate a crime scene now.
Any interesting question that arises here is that, do identical twins have the same DNA fingerprint?
To a standard DNA test, identical twins are indistinguishable- since they come from the same fertilized egg. However, there is one feature that sets them apart- their fingerprint. Fingerprints are influenced by factors like exposure to amniotic fluid, pressures to the womb etc. Their fingerprint makes them different.
In modern times, the DNA fingerprinting technique has been modified due to the presence of advanced technology.
These days, microsatellites or short tandem repeats (STRs) are used for profiling. They are 5-6 base pair long and so only a small amount of DNA sample is needed. Several copies of STRs are created using Polymerase Chain Reaction (PCR). In this technique, small DNA fragments called primers bind to desired complementary sequences to mark the starting point of copying the DNA. In STR analysis, fluorescent tagged primers attach to either end of the STR of interest. Gel electrophoresis is then used to separate the DNA fragments according to their size.
A particular pattern is obtained on the screen when each fragment passes by a laser. The laser causes the fragments with fluorescent tags to glow with a specific colour. The output is displayed as a series of colourful peaks highlighting the colour and length of each STR sequence.
It is funny to think of a time when DNA fingerprinting was not available, we take it for granted these days. It has indeed revolutionized the field of biotechnology and forensic science since it’s invention in 1984.