Workshop on RFLP and RAPD

 

St. Joseph’s College for Women (A), Visakhapatnam

 DEPARTMENT OF BIOCHEMISTRY

&

Internal Quality Assurance cell (IQAC)

 

 

Organized A

2 Day Workshop on RFLP & RAPD

In collaboration with

SHRIJOSHIKA MOLECULAR DIAGNOSTICS AND RESEARCH CENTER, VIZAG

14^th and 15^th March, 2023

 Venue- Shrijoshika Molecular Diagnostics lab, Near Lions Club, Visakhapatnam.

 

 

 

 

 

 

St. Joseph’s College for Women (A), Visakhapatnam

Department Of Biochemistry & Internal Quality Assurance Cell

 2 Day Workshop on RFLP & RAPD

In collaboration with

SHRIJOSHIKA MOLECULAR DIAGNOSTICS AND RESEARCH CENTER, VIZAG

14^th and 15^th March, 2023                                                                Time: 9 AM to 4 PM

 

Day 1 session
9.30 am	Welcoming students and welcoming resource speakers	Dr P. Mary Anupama
9.35 am	Prayer song	3rd  BBC team
9.40 am	Rationale	Dr P. Mary Anupama
9.45 am	Introduction to Resource speakers	Dr Mousami Shankar A
9.50 am	Dr Jagadeesh and Dr Kalyani, Directors	General introduction
10.00 am	Session 1 of RFLP Batch 1
Lunch Break From 12.15 to 1.0 pm
1.0 pm	RAPD session for batch 1
4.0 pm	Close of first day program
DAY 2
9.15 am	Introduction to RFLP session 1 to batch 2
12.00 noon	Session 2 of RAPD for batch 2
1.0 to 1.30 pm	Lunch break
4.0 pm	Certificate distribution
5.0 pm	National anthem
2.40 pm 4.0 pm	Experiment continues        Certificate distribution

 

 

 

 

Rationale for Workshop on RFLP & RAPD

Dates- 14^th and 15^th March, 2023

Random Amplified Polymorphic DNA (RAPD):

RAPD was developed by Welsh and McClelland along with Williams in 1990.

It is pronounced as ‘rapid’.

It is based on PCR assay and it doesn’t need require any prior sequencing of DNA.

This procedure uses short arbitrary primer of 8-12 bp that randomly amplifies the region of DNA.

This primer serves as both forward and reverse primer.

This reaction proceeds when a single primer anneals to the genomic DNA at two distinct sites on the complementary strand of DNA template.

The amplification of segment of DNA depends on the positions complimentary to the primers’ sequence.

The fragments obtained from RAPD are between 0.2 to 5.0kb and can be viewed by using agarose gel electrophoresis stained by ethidium bromide or with the help of polyacrylamide gel electrophoresis.

If any mutation occurs in the primer binding region then no any PCR product will be produced, yielding a distinct pattern of amplified DNA segments on the gel.

Application:

Distinct pattern of amplification is seen in different samples. This is why RAPD can be used for studying polymorphism.

RAPD is applicable for the mapping of genome, analyzing linkage, and individual specific genotyping.

RAPD markers are dominant in nature so it has restrictions for mapping purpose.

RAPD is strictly laboratory dependent so it requires sensitivity.

Restriction Fragment Length Polymorphism (RFLP):

It was one of the first methods used for the analysis of DNA in various fields such as forensic science.

It is a hybridization based technique.

It was invented by Alec Jeffreys, an English scientist in 1984 during his research in genetic diseases.

RFLP uses particular restriction endonuclease enzymes that cuts at its specific site yielding fragments of various lengths along with the fragment of interest.

The length of the distinct fragments is determined by using blotting, now replaced with sequencing.

RFLP markers are largely locus-specific and are co-dominant in nature due to the nature of restriction endonuclease used.

Steps for RFLP are as follows:

DNA extraction is done from saliva, blood or other samples and is purified.

Restriction endonucleases digests the purified DNA resulting restriction fragments.

Now the restriction fragments are examined using gel electrophoresis.

The gel is now treated with luminescent dyes for the visibility of DNA bands.

 

Applications:

RFLP was one of the first techniques applied for genetic fingerprinting/profiling.

It is used for identification of inherited diseases, carrier of that diseases, genetic mapping, and heterozygous detection.

The molecular basis of the RFLP is that any point mutations as such deletions, substitutions and insertions or alterations like duplications, inversions within the genome can eliminate or form new restriction sites. These alterations in genome can be detected by analyzing fragments of variable length, digested with restriction endonuclease enzyme