DNA Fingerprinting Procedure , Advantage & Application | Biology

                                   DNA Fingerprinting

Good day, biology enthusiasts!

In this blog, we're diving into one of the most groundbreaking advancements in forensic science — DNA Fingerprinting. But before we explore its application, let’s understand what it actually means.

What is DNA Fingerprinting?

DNA Fingerprinting is a technique used to identify individuals based on their unique DNA profile. Interestingly, about 99.9% of the DNA in every human being is identical. However, it's the remaining 0.1% that makes every individual genetically unique.

This uniqueness is determined by sequences called VNTRs – Variable Number of Tandem Repeats. VNTRs are short sequences of DNA that repeat multiple times in a row, and the number of repeats varies from person to person.

Let’s take an example:

• Person A’s DNA: ATGC

• Person B’s DNA: ATTGC

The extra ‘T’ in Person B's sequence represents a variation in the VNTR, and it’s these small differences that allow scientists to distinguish between individuals.

🧪 How is a DNA Sample Collected?

To create a DNA fingerprint, a sample containing DNA (like blood, saliva, or hair) is collected from a suspect or individual. This sample is then processed and compared to DNA found at a crime scene or from another source.

This technique was pioneered by Sir Alec Jeffreys, a British geneticist, in 1984. He discovered that every individual carries a unique pattern of minisatellites — which laid the foundation of modern DNA fingerprinting.

 Procedure  -  A DNA fingerprint is the same for every cell, tissue, and organ of an organism. You can not be altered by any known treatment. This is a laboratory procedure consist of following steps -

Isolation Of DNA - The DNA  is isolated in a small amount from victims of blood cells or hair.

Cutting, Sizing and Sorting Of DNA - Restriction enzymes are used to cut the DNA at specific places. If there is a change in the cutting site then it will stop the enzyme from cutting the DNA. Thus a very small difference in the DNA of two individuals can result in the cut pieces of DNA of different sizes and in turn cause the fingerprints of the two individuals to differ.

Electrophoresis - The DNA is separated according to size by gel electrophoresis. Electrophoresis is a laboratory procedure in which DNA pieces are sorted by size. In this method, the DNA was loaded into the well and electric current is applied. The negative charge DNA move towards positive charge cathode through the gel. The small DNA fragments move faster than large DNA fragments. Now the electric current switch off and in this way, the DNA fragments are separated through electrophoresis.

Transfer Of DNA - The DNA pieces are transferred to the nylon sheet by placing agarose gel and nylon sheet next to each other overnight.

Probing - Probe is a fragment of DNA that can be radioactively labeled. The DNA fingerprinting is generated by adding a tagged probe to the nylon sheet. The probe only attaches to the pieces of DNA that are complementary.

DNA Fingerprints - Now this probe which is tagged is visualized by exposing the nylon membrane to the  X-Ray film. When developed, the resultant visible pattern is the DNA fingerprint looks like a bar code in the grocery counter.

Advantage Of DNA Fingerprinting -

• DNA fingerprints can not be altered by any method.
• Every individual has its own unique DNA fingerprints.
• DNA fingerprints can not be duplicated.
• It is a very reliable technique for the identification of an individual. 

Drawbacks Of Fingerprinting -

• The cost of testing is very expensive.
• Error in hybridization or probing will give wrong results.
• If there is contamination in the sample then results also affected. 

Application Of DNA Fingerprinting -

• Forensic Science - DNA fingerprinting is very helpful in detecting criminal cases like murder, robbery, rape, kidnapping, etc.
  
  
• Diagnosis Of Paternity and Maternity - DNA profiling can be used to identify whether the two people are related to one another. Thus is helpful for solving paternity or maternity disputes.
  
  
• Diagnosis Of Diseases -  DNA profiling is used in detecting genetic disorders caused in the body.
  
  
• Protection Of Legal Right -  DNA fingerprinting is used to legally protect new varieties of plants and animals.
  
  
• Identification Of Exchange Child - In hospitals some times baby are exchanged. DNA testing helps in the identification of real children.

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Western Blotting - Principle , Method & Diagram

                                      Western Blotting

Western blotting is a type of blotting which is used for identifying particular protein from a sample by using an antibody specific to that protein. This technique was developed by Towbin and his co-worker in 1979. This technique is also known by another name Immunoblot. In this method nitrocellulose and nylon, the membrane is used for blotting. Sometime Polyvinylidene fluoride is also used for blotting purposes. The probe used is an antibody to detect a specific protein. The positive signal indicates that the protein of interest is present in the given sample. The antibody is labeled with radioactive material so that it produces light after combining it with the enzymes.


Detection -  Western blotting can be detected in two ways -

Direct Detection -  Primary antibody which is made up of purifying the desired protein and then injecting the protein into an animal is used for detecting an antigen on the blot.  The primary antibody is labeled with an enzyme or fluorescent dye.

Indirect Method - Here primary antibody is added to the antigen which is followed by a labeled secondary antibody direct against the primary antibody. This antibody is made by injecting the primary antibody into a different animal species.

Related Link - Southern blot technique 

The procedure of western blotting -  Western blotting is consisting of the following procedure -

• Gel Electrophoresis -  The sample protein is separate out according to size on a gel using SDS- PAGE.

• Transfer to membrane - Transfer of protein from polyacrylamide gel to nitrocellulose is done by applying an electric field for 2 hours. After some time the protein is bound to a nitrocellulose filter.

• Blocking -  To prevent nonspecific protein interaction, the membrane is blocked. Blocking is done by placing the membrane in the solution of BSA ( Bovine Serum Albumin).

• Adding Antibody -   The primary antibody solution is added to the membrane then washed to remove unbound antibody after which the secondary antibody is added which adhere to the primary antibody. The membrane again washes to remove unbound antibody. The secondary antibody linked to the enzyme for which substrate is added which produces color helps in the detection of the protein of interest.

• Detection - The radioactive specific antibody is added on such a membrane. The antibody is labeled with the radioactive isotope.

Application Of Western Blotting -

• This technique is used to determine whether a gene is expressed or not.
  
  
• Protein-Protein interaction can also be studied with the help of this technique.
  
  
• It is also used for diagnosing several medical tests like viral, genetic, and parasitic diseases.
  
  
• HIV detection can be possible by this technique.
  
  
• It is useful in an autoimmune disorder.  

Electrophoresis

                                      Southern Blotting

In molecular biology there are three techniques used for detecting DNA / RNA /protein ,  Southern blotting for DNA detection , Northern blotting for RNA detection and Western blotting for  detecting protein .  The term blotting is generally refers to the transferring of the   DNA /RNA / Protein into carrier like nylon membrane .  After blotting the transferred molecules are visualized by autoradiography , chromogenic detection method , silver staining method etc . This technique was developed by  Edwin Southern in 1975 .

Procedure -

• The DNA is isolated from the cell of interest .
• Now the DNA is digested by restriction enzyme . Restriction enzyme cut the DNA molecules into desired fragments .
• The fragments are separated by agrose  gel electrophoresis into different size .
• The double stranded DNA  is denature into single strands by sodium hydroxide .
• The DNA fragments are transferred onto  membrane by capillary action . There are two type of  membrane are used in this method - nitrocellulose membrane and nylon membrane .
• The small segments known as probes is labelled with either with radioactive chemical or fluorescent dye  for identification . 
• 
  The probe will bind strongly to the sequence and lead to the formation of double stranded DNA .
• The unbound molecules are removed by washing process .

Applications Of Southern Blotting -

• Southern Blotting is used for detecting particular gene in the genome .
• It is used for gene cloning .
• It determine the map position of DNA sequence in the chromosome .