Restriction Fragment Length Polymorphisms
(RFLPs)
Dr. Hani Alhadrami
[email protected] www.hanialhadrami.kau.edu.sa
Outlines
What is polymorphism?
Example of polymorphism (SNPs).
What is RFLP?
Restriction endonuclease.
Classification of restriction endonuclease.
General Applications of RFLP.
Case study to use RFLP in paternity cases.
Case study to use RFLP in the detection of disease and mutation.
Polymorphism
Natural variations in a gene, DNA sequence or chromosome that have no adverse effects on the individual and occur with high frequency in the general population.
Example of polymorphism is a
single nucleotide polymorphism
(SNP).
SNP
Is a variation in a DNA
sequence occurs when a single nucleotide (i.e. A,T,C or G) in the genome differs between members of a biological
species or paired of
chromosomes in human.
Example: two sequenced DNA fragments “AAGCCTA” &
“AAGCTTA” from different individuals, contain
difference in a single nucleotide.
SNPs
SNPs occur normally throughout an individual's DNA.
They occur once in every 300
nucleotides (meaning that there are 10 million SNPs in the human genome).
SNPs can act as biological markers to help scientists locate genes that are
associated with disease, and they play a role in predicting an individual’s
response to certain drugs and track
the inheritance of disease genes within families.
SNPs have no effect on health or development.
RFLP
Restriction Fragment Length Polymorphism (RFLP) is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their DNA by restriction enzymes (restriction
endonuclease).
In RFLP technique, the DNA sample is broken into pieces (digested) by restriction enzymes and the
resulting restriction fragments are separated according to their length by gel electrophoresis. .
The similarity of the generated patterns can be used to differentiate species (and even strains) from one
another.
Restriction Endonuclease
Restriction endonuclease: an enzyme obtained from bacteria that cuts DNA into specific short segment.
Purified restriction endonuclease allow the molecular biologist to cut DNA molecules in a precise and
reproducible manner.
The discovery of these enzymes, which led to Nobel
Prizes for W. Aber, H. Smith and D. Nathans in 1978, was one of the breakthroughs in the development of genetic engineering.
Principle of Restriction Endonuclease
Restriction occurs because the bacterium produces an enzyme that
degrades the phage DNA before it has time to replicate and synthesis of new phage particles (a phenomenon known as host-controlled
restriction).
Bacterium’s own DNA, (the destruction of which would be lethal), is protected from attach because it carries additional methyl groups that
Classification of Restriction Endonuclease
Restriction endonuclease are classified based upon their mode of action into three different classes Types I, II & III.
Types I & III are complex and have a limited role in genetic engineering.
Types II are the most important in genetic engineering and gene cloning.
The central feature of type II endonuclease is that each enzyme has a specific recognition sequence at which it cuts a DNA
molecule and nowhere else.
For example: restriction endonuclease called PvuI (isolated from Proteus vulgaris) cuts DNA only at hexanucleotide CGATCG.
Whereas, a second enzyme from the same bacterium called PvuII cuts at different hexanucleotide CAGCTG.
Blunt ends & Sticky
Blunt ends or flush ends:
-N-N-A-G-C-T-N-N- -N-N-A-G C-T-N-N- -N-N-T-C-G-A-N-N- -N-N-T-C G-A-N-N-
Sticky ends or cohesive end:
-N-N-G-A-A-T-T-C-N-N- -N-N-G A-A-T-T-C-N-N- -N-N-C-T-T-A-A-G-N-N- -N-N-C-T-T-A-A G-N-N-
AluI
EcoRI
Sticky ends
“N” = A, G, C, or T
Blunt ends & Sticky
Blunt ends or flush ends:
It occurs when restriction endonuclease makes a simple double-strand cut in the middle of the recognition
sequence.
Sticky ends or cohesive end:
The two DNA strands are not cut at exactly the same position so that the resulting DNA fragments have short single-
stranded overhangs at each end. These are called sticky ends as base pairing between them can stick the DNA molecule
back together again.
Applications of RFLP
There are several applications for RFLP, for instance:
1. It can be used as a vital tool in genome mapping and genetic disease analysis.
2. It is useful in Genetic Fingerprinting and the
identification of samples retrieved from crime scenes (forensic applications).
3. It has a role in the determination of paternity cases and the source of a DNA sample.
4. It can be used to determine the disease status of an individual (i.e. mutations detection).
Case Study I - A Paternity Case Scenario - I
RFLP technology to determine if Jack is the father of Jill's child named Payle.
In this scenario, DNA was extracted from white blood cells from all three individuals and subjected to RFLP analysis. The results are shown below:
In this case, it appears that Jack could be the father, since Payle inherited the 12.4 kb fragment from Jill and the 4.3 fragment from Jack. However, it is possible that another man with similar RFLP pattern could be as well.To be certain, several more RFLP loci would be tested. It would be highly unlikely that two men (other than identical twins)
Case Study I - A Paternity Case Scenario - II
RFLP technology to determine if Jack is the father of Jill's child named Payle.
In a different scenario, DNA was extracted from white blood cells from all three individuals and subjected to RFLP analysis. The results are shown below:
This time, it can be determined that Jack is NOT the father of Payle since Payle has a band of about 6 kb and Jack does not. Therefore, it is very probable that Payle's father is not Jack, though it is possible that Payle carries a new mutation at this locus and a different sized band was produced.
Case Study II - Detection of Disease
RFLP technology to determine if a person carries any Cystic Fibrosis (CF) alleles.
Anyone with CF must be homozygous for disease alleles. From pedigree information, we can often determine who in this family is a carrier. However, if a couple comes to a genetic counsellors, often an RFLP analysis is performed on the couple's DNA.
RFLPs are known for CF and so it would be easy to determine if a person were homozygous wild-type (wt), heterozygous "carrier", or homozygous disease alleles and thus have CF (fig A).
For couples expecting a child, it would be simple to test both parents and make a prediction about the eventual disease status of their fetus. For example, if both parents were homozygous (wt), then all of their children would also be homozygous (wt): (fig B)
Case Study II - Detection of Disease
RFLP technology to determine if a person carries any Cystic Fibrosis (CF) alleles.
However, if both parents were heterozygous, they could have children with any of the three genotypes, though
heterozygous children would be twice as likely as either of the homozygous genotypes
