Restriction fragment length polymorphism (RFLP) marker
Before starting to RFLP, note that marker means not a particular things or individual something or a big something... it may be Dna sequence or may be a polymorphism about Dna fragments from which we can understand or detect or diagnose something related to whatever we want to study... ok fine ! Now lets starts towards RFLP concept !
Restriction fragment length polymorphism (RFLP)
Concept of RFLP
In this method DNA is digested with restriction enzyme(s) that cuts the DNA at specific sequences, electrophoresed, blotted on a membrane and probed with a labeled clone.
▶️Polymorphism in the hybridization pattern is revealed and attributed to sequence difference between individuals.
▶️The DNA sequence variation detected by this method was termed restriction fragment length polymorphism (Botstein et al., 1980).
▶️Grodzicker first described this phenomenon for mutant strains of adenovirus.
▶️Botstein, White, Skolnick and Davis realized that RFLPs were not necessarily associated with specific genes but were distributed throughout the genome, and they made a detailed proposal for using RFLPs to map all the genes in the human genome.
▶️RFLP is the most widely used hybridization-based molecular marker.
▶️RFLP markers were first used in 1975 to identify DNA sequence polymorphisms for genetic mapping.
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[Point to Remember]
✅️Variation in one DNA fragment obtained with a specific enzyme is treated as one RFLP
✅️In experiments we performe may be in many fragments variation can occur in comparison with reference sequence so we called them ( such many variations ) many RFLP
How / why RFLP Operate/Occur or we use RFLP / Which conditions tends to use RFLP technique..( RFLP means just a fragments variation seen with the help of autoradiography )?
Answer ➡️
▶️RFLP was the very first technology employed for the detection of polymorphism, based on the DNA sequence differences. RFLP is mainly based on the altered restriction enzyme sites, as a result of mutations and recombinations of genomic DNA.
▶️RFLPS arise because mutations can create or destroy the sites recognized by specific restriction enzymes, leading to variations between individuals in the length of restriction fragments produced from identical regions of the genome.
▶️Although two individuals of the same species have almost identical genomes, they will always differ at a few nucleotides due to point mutation and insertion/deletion.
▶️Some of the differences in DNA sequences at the restriction sites can result in the gain, loss or relocation of a restriction site.
▶️To identity such differences we use RFLP technique.
RFLP Procedure
RFLP analysis comprises the following basic steps:
- DNA isolation.
- Cutting DNA into smaller fragments using restriction enzyme(s).
- Separation of DNA fragments by gel Electrophoresis.
- Transferring DNA fragments to a nylon or nitrocellulose membrane filter [Southern Blotting]
- Probe binding [Hybridization]
- Washing
- Exposure to X ray film
- Visualization of specific DNA fragments using labeled probes.
- Analysis of results.
We can understand RFLP by following example
▶️Based on the presence of restriction sites, DNA fragments of different lengths can be generated by using different restriction enzymes.
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| A schematic representation of restriction fragment length polymorphism (RFLP) analysis as molecular marker. |
▶️In above diagram two DNA molecules from two plants (A and B) are shown.
▶️ln plant A a mutations has occurred leading to the loss of restriction site that can be digested by EcoRI.
▶️The result comes that when the DNA molecules are digested by the enzyme Hindlll, there is no difference in the DNA fragnrents separated.
▶️But with the enzyme EcoRl, plant A DNA molecules is not digested & plant B DNA molecule is digested.
▶️This practice results in a polymorphic pattern of separation
[You can also follow this example for understanding]
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| Restrictions fragment length polymorphism |
You have one question that how we know or detect variation from this example... so see,
[Sample 1]
In above diagram There is wild type restriction site ( i.e Ecoli restrictions site means Ecoli cut to this site ) and after cutting we get to fragments.
[Sample 2]
In above diagram, to your right side there is mutant restriction site that means A - T changes to C - G this results in mutant site & Overall site is now not for Ecoli therefore Ecoli cannot cut it. So like sample 1 two fragments cannot obtained.
[So What Happen]
And electrophoresis graph of sample 1 & 2 not showing similar pattern of fragment so there is distinguishable polymorphism i.e many forms or different forms.
But if there will be similarity so no polymorphism occur and the conditions are normal. So therefore marker work only in polymorphic condition.
[Conclusions]
▶️A single base change within a restriction site is a readily detectable genetic marker because the mutated site is no longer cleaved by the enzyme in question.
▶️Two chromosomes that differ by such a mutation are then distinguishable on the basis of a restriction fragment length polymorphism (RFLP), which arises because a particular cleavage site is present in only one of the two DNA molecules.
▶️A mutation that gives rise to an RFLP, thus represents a genetic marker.
▶️RFLPS have only two alleles: the site is present or absent.
▶️The maximum heterozygosity is 0.5.
▶️The RFLP markers are codominantly inherited and highly reproducible.
[References]
Chawla, H. S. Introduction to plant biotechnology . 3rd ed., Science Publishers, 2009, pp. 356-358.
Kumar, Pranav , and Praveen Verma. Biotechnology A problem approach . 5th ed., Pathfinder publication New Delhi, india, 2019, pp. 860-864.
Jiang, Guo-Liang. "Molecular Markers and Marker-Assisted Breeding in Plants ." https://www.intechopen.com/chapters/40178. 22 May 2013, 10.5772/52583.
Mishra, Sidharth, et al. "Improvement of Molecular Markers in Animal Science." Advances in Veterinary Sciences, vol. 2, 2019, pp. 93-129, www.researchgate.net/figure/Restriction-fragment-length-polymorphism-RFLP-procedure-1-Extract-DNA-from_fig1_337589428.
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