If you think that your DNA only contains characteristically genetic information from generation by generation, you are wrong. Your DNA makes you unique from others. 

But how?

Well, here is the answer.

The human genome contains specific sequences called polymorphic regions.

These regions are commonly found in the “Intron” (non-coding sequence of DNA) part of DNA. These polymorphic regions differentiate between two individuals called polymorphism or variation. 99.9% of the population contains the same DNA but only 0.01% varies between individuals.

These sequences are repetitive, non-coding, junk sequences. 

Figure 1- STR alleles found in the intron (non-coding part) part of DNA

Brief History of DNA Profiling

The year 1984 may be considered as an unfortunate year for criminals when a British Geneticist named Alec Jeffreys developed a technique known as DNA fingerprinting to identify different individuals.

From 1986 DNA fingerprinting was widely used in the field of forensic science and criminal investigation. 

He develops the technique known as VNTR (Variable Number of Tandem Repeats). A long specific sequence is present on satellite DNA having a repeat of nucleotides.

After VNTR other techniques were developed for DNA profiling like RFLP (Restriction Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA, etc.

Nowadays recent advanced techniques focus on short nucleotide sequences i.e., 2-5 bp long, which shows polymorphism (variations) in individuals. 

Why STR?

The nucleotides (ACGT) present on DNA show a repetitive arrangement in specific regions.

This repetitive sequence is called a Short Tandem Repeat or STR. STR sequences are 2 to 5 base pairs in length has a repetitive sequence like “-AGAGAGAGAG-”. Sometimes it shows tetrameric repeats.

For example, the 16bp sequence of “-AGATAGATAGATAGAT-” would represent 4 copies of tetramer “AGAT”. This repetitive sequence would not same for two individuals.

Nowadays STRs become the backbone of the DNA profiling system. STR markers are co-dominant markers. STR typing is a comparatively easy, reliable, less time-consuming process.

STR typing always gives a positive result in Criminal activities such as victim-suspect identification, paternity disputes in kidnapping or lost child case, Disaster victim cases, etc. 

How Does STR Works?

For example, a 3 years old child is lost to his parents.

The child was recovered by police after 7 days. On the next day, a couple goes to the police station and claim that they are the real parent of the child.

Now, STR will decide whether they are the real parent of the child or not.

As we know STRs are co-dominant markers, which means an offspring receives both STR alleles from father and mother. It means a perfect matching of 50% with both parents.

Now, if the report of STR typing shows the child contains the same repetitive sequence from both parents it means the child belongs to the couple who claimed him.

What Evidence is Needed for STR Typing?

Any biological evidence which contains DNA like Blood, saliva, hair (with root), bone, teeth, skin cells, tissue samples, etc.

How STR Typing is Done?

There is kit-based STR profiling available. Generally, 3 types of kits are available in the market. 

1. Identifier/ Global Filer Kit: To identify paternal maternity (when both parents are available). Contains 27 primers.

2. Y- Filer Kit: To identify paternity (only father is available). Contains 23 primers. 

3. Minifiler Kit: (use for Disaster victims’ identification or in case of the degraded sample)

Steps of STR Profiling

STR profiling is commonly divided into 4 steps:

1. Extraction

This is the first stage of STR profiling. First, the DNA has to be extracted from the sample. Samples can be any biological fluid like blood, saliva, or hair (with root), teeth, bones, skin cells, tissue, etc. 

If the biological fluid like blood and saliva is present on cloth or any object then firstly cut it and dissolve it into a mixture of G2 buffer (lysis buffer), Detergent like SLS (sodium laurel sulfate), proteinase K enzyme, etc. The blood cell lysis happens in this mixture and the nucleus DNA comes out in the solution. 

Now, there are major 2 methods available for DNA extraction 

a) Organic Extraction Method: Phenol- Chloroform method, Chalax-100 methods, etc. are available. Organic methods are time taking. The process of organic extraction is given in Figure 2.     

Figure 2 - Organic DNA Extraction

b) Automated DNA Extraction System: This is a magnetic extraction system. Biorobot DNA extraction system is an advanced technique that can extract the maximum quantity of DNA from a degraded sample. Image 4 is describing the extraction procedure by Bio-robot.

Figure 3 - DNA Extraction By the Automated Magnetic Extraction 

2. Quantitation

Fluorescence-based kits are also available to measure the quantity of DNA. Gel electrophoresis is used for this purpose. Bromophenol is used as a fluorescence dye. DNA fragments were checked under UV light to measure quantity and quality.

3. Amplification

The major step of STR profiling. This step needs Polymerase Chain Reaction or Thermocycler. As described earlier, STRs are the short repetitive sequence that individualizes people. We need maximum copies of the STR regions and PCR makes copies of STR regions. 

Here, a DNA expert needs to follow some major steps to make copies of STR regions by PCR. 

Commercial kits are available now. These kits contain mastered mixture of Primer, dNTPs (oligonucleotide), DNA polymerase, buffer.

Primer: 15-18 bp size short pre-made DNA sequence, bind before the STR regions need to copy. 15-18 bp primer is considered a standard size primer. If primer size is less than 15-18 bp it binds in any region of DNA and if the size is more than 15-18 bp the primer will take more time to bind. In both cases, we will get a negative result.

dNTPs: Deoxyribose nucleotide triphosphate i.e., dATPs, dTTPs, dGTPs, dCTPs. Binds with STR’s complementary sequence from where the primer is bound. 

DNA polymerase: “Taq polymerase” is a specific DNA-dependent DNA polymerase that elongates the nucleotide chain with its complementary sequence. 

Buffer: Mg2+, G2 buffer used to remove contamination, and maintain pH.

Figure 4 - Mastered Mixture

How PCR Works?

Now, all the mixture is kept in the PCR along with the “Templet DNA” (DNA needs to be replicated). Here, 3 steps were added.

First, Denaturation: DNA needs to denature first. Double-stranded DNA breaks and appears into single Stranded DNA. This procedure is done at a temperature of 94-980 C.

Second, Annealing: Temperature needs to be low in this step. A maximum temperature of 50-560 C needs to complete the annealing of DNA. Primer binds with the complementary sequence of nucleotides present before the STR region.

Third, Elongation: Now the temperature of PCR needs to be high again. Maximum 720C is needed to complete this step. Taq polymerase is attached with the strands of DNA and completes the joining of nucleotides with their complementary sequence. 

PCR reaction is also known as Thermocycler because a cyclic reaction is happened by changes in temperature.

4. Capillary Electrophoresis/Sequencer

First, fluorescence-labeled pre-made copies of DNA are poured into the tube and loaded into the sequencer.

When a capillary array is filled with a column ER and placed into the sample wells, an electric current is created, and DNA fragments are pushed kinetically into the capillaries near the ends of the capillary array.

The array is placed in the buffers for the cathode and anode. To control sample migration across the capillary array, the instrument uses a certain voltage and temperature.

The fragments are separated by size. The smallest fragments travel the fastest, and the final stage is detection, which occurs when fluorescently labeled particles pass through the detection window, causing the fluorescent dyes to be excited by the laser.

A CCD camera records the emitted light after it has been spectrally separated. Each fragment's fluoresce signature is being captured.

The raw data is supplied to the collection software as a peak and is now ready to be analyzed with a specialized software program.

Figure 6 - Capillary Electrophoresis Step By Step

Figure 7 - Graph/ Peaks Generates After Sequencing

Here are we done STR typing. Sometimes useless sequences of your DNA make you unique. Would you still call it junk????

Written by: Soumajit Adhikary