What is DNA Fingerprinting?

The genetic makeup of living organisms is shown by a technique known as DNA fingerprinting. The difference is the satellite region of DNA is shown by this process. Alex Jeffreys has invented the process of DNA fingerprinting in 1985. Any biological samples such as blood, hair, saliva, semen can be used for DNA fingerprinting. DNA fingerprinting is also known as DNA profiling or molecular fingerprinting.

Various Steps of DNA fingerprinting:

Isolation of DNA

From cells or tissue, DNA samples are recovered, or sometimes from bacterial cell DNA is obtained. A small amount of DNA samples is needed for isolating DNA. The isolated DNA samples are now subjected to the digestion of DNA. Bacterium divides by binary fission.

Digestion of DNA by Restriction Endonuclease Enzyme

Using restriction enzymes, DNA is cut into fragments. DNA is cut at a specific position by the enzyme. Restriction fragments are those sequence of DNA which are cut by using a restriction enzyme. Thousands of DNA fragments are obtained by this process.

Restriction enzyme: Restriction endonuclease is a restriction enzyme. Restriction enzyme cuts the DNA at a specific position. A restriction enzyme is a bacterial enzyme. dsDNA is cut into fragments after recognizing specific nucleotides.

Electrophoretic Separation of Different Fragments

Based on shape and size, the separation of the fragment is done. The DNA fragments are injected into the wells, and along with it, the current is applied. It is attracted to a positive end as it is negatively charged. The rate of movement of the shorter fragment is faster than, the longer fragment. Based on the size, DNA is separated.

Transfer of DNA On Nylon/Nitrocellulose Membrane

On a nylon sheet, DNA fragments are transferred. Then, it is soaked overnight through the process of southern blotting.

1. Probe Labelling : To the nylon sheets, colored probes are added, which are complementary to the target sequence. The probe sticks on the sheet at one or more than one specific place.
2. Hybridization: In this process, probe DNA is hybridized with a complementary sequence present on a nylon membrane. Now washing of membrane is done to remove the non-specific binding and for a background clearance.
3. Autoradiography : For detecting the sequence bounded in the genome with the probe on the membrane, autoradiography is done. In this, the hybridized membrane is aligned with an X-ray film. In X-ray film, it looks like a barcode, and it is known as DNA fingerprints.
4. Interpretation of Band Pattern : Different software is available on computers that interpret the band pattern. The position of bands is compared.

Uses

• It is used in the diagnosis of inherited disorders.
• It is used for determining child paternity and maternity.
• It is used for personal identification.

Gel Electrophoresis

The separation of charged molecules on the application of electric field is known as Electrophoresis. On several factors, the mobility of individual molecules is based. Those factors are net charge, charge/mass ratio, molecular shape. Macromolecules such as DNA, RNA, and proteins are separated based on shape and size.

Principle

An electric field is involved in this process. On it, small pores are present in which the sample is put. In it, pores are present through which molecules travel. The molecule, which is smaller in size, travels more distance through it. The negatively charged molecules are DNA or RNA. When protein is subjected to this process, it is first mixed with sodium dodecyl sulfate. Then, the protein is unfolded into linear shapes by treating this detergent and gets coated with a negative charge.

It is of two types;

1. Agarose
2. Polyacrylamide Gel Electrophoresis (PAGE)

Agarose

 It is poured vertically. This process separates large molecules. By this, large DNA molecules are separated. Ethidium bromide is used first. This agar is used as a support medium. Cellulose acetate is more expensive. It is an acidic polysaccharide; a monomer of sulfated galactose is present in it. In a buffer, the gel is prepared, and after its preparation, it is spread over a microscopic slide. Its extraction is done from seaweed. It is derived from agarose. When added to boiling, liquid gets dissolved. On lowering the temperature, the agarose solidifies. By adjusting its concentration, the size of the pore is determined. It is mainly hydrocolloids therefore delicate in nature. When molecules to be separated are protein, protein complexes, weak bonds are present in them, such as hydrogen and hydrophobic bond.

Advantage :It is easy to prepare. For its preparation, only a small concentration is required. The resolution power of it is better than filter paper. A high quantity of protein can be separated.

Disadvantage : High electro-osmosis is seen. Less resolution is seen as compared to polyacrylamide gel.

Application

In immunoelectrophoresis, it is widely used. Different protein mixtures are separated by using this.

Polyacrylamide Gel Electrophoresis (PAGE)

In this, it is poured horizontally. This separates small molecules. Coomassie blue is generally used in this. Preparation of polyacrylamide is done by polymerizing acrylamide monomers. Covalent cross-links are present in polyacrylamide gel. Polyacrylamide is harder. It is thermostable, transparent, strong, and inert. The separation of protein is done based on the charge to mass ratio.

Types of PAGE

According to the separation, condition PAGE is divided into;

• NATIVE-PAGE and Denatured PAGE

NATIVE PAGE: In non-denaturing conditions, NATIVE PAGE is run. On charge, size and shape separation is based. A mixture of protein is purified and separated by using NATIVE PAGE.

• DENATURED PAGE-SDS PAGE

On the molecular weight of protein, its separation is based. The molecular weight of the protein is determined by using this SDS-PAGE. This sample checks the purity of the protein sample.

SDS PAGE is also known as sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This SDS PAGE is widely used in biochemistry, forensics, and molecular biology. SDS is a detergent, and when it is added to PAGE, it is known as SDS-PAGE. Protein molecules are coated by SDS, resulting in a constant charge-mass ratio. Since SDS is an anionic detergent, its role is to denature the secondary and tertiary linkage through wrapping all around the protein. The negative charge present on SDS destroys the complex structure of the protein.

The required material is a sample, Ethidium bromide, gel loading dye, TBE, and autoclave.

Context and Application

This topic is important for many entrance exams

• Bachelors of Science (botany and zoology)
• Masters of Science (botany and zoology)