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fragment length
Restriction Enzyme is one of a class of
enzymes that cleave DNA at specific base
sequences; produced by bacteria to degrade
foreign DNA.
The mutation can either make a new
restriction site or remove a site that already
Accordingly In that bases you will be able to
detect these types of polymorphisms by RFLP
Came with each Restriction Enzyme a data
sheet which contains too many important
information about the properties of the
enzyme , the best buffer , the right
temperature , unit definition etc.
As its shown in this Data sheet for example
the enzyme name is NdeI and the best buffer
for it is V5 , you should incubate your digested
mixture at 37c for at least two hours and the
enzyme concentration is 5units/l.
Also its shown that in the data sheet what we
call the unit definition: 1 unit is sufficient to
digest 1g from lambda DNA (or PCR product).
As long as the enzyme concentration is
5units/l and you want to work with 1 unit/l
therefore you have to take 1/5 l from the
stock enzyme (0.2l) or you could make a
dilution 1:5 ratio and then take 1l from the
diluted enzyme.
Take 2l from buffer v5.
According to the sharpness of your PCR
product take minimum 5 l.
Complete your volume to 1o by adding
sufficient amount of DH2O.
In our example suppose you have a sharp
band in your PCR so our RFLP mixture would
1. 2l buffer V5.
2. 0.2l NdeI enzyme.
3. 5l PCR product.
4. 2.8l DH2O.
Incubate the mixture at 37c overnight , then
in the following day prepare a right
percentage of an agarose gel (depending on
expected band sizes).
Lastly after running your gel into the
electrophoresis apparatus check into UV light
illuminator or Gel documentation system.
Analysis of RFLP variation in genomes was a vital tool
in genome mapping and genetic disease analysis.
If researchers were trying to initially determine the
chromosomal location of a particular disease gene,
they would analyze the DNA of members of a family
afflicted by the disease, and look for RFLP alleles that
show a similar pattern of inheritance as that of the
Once a disease gene was localized, RFLP analysis of
other families could reveal who was at risk for the
disease, or who was likely to be acarrierof the
mutant genes.
RFLP analysis was also the basis for early
methods ofGenetic fingerprinting , useful in
the identification of samples retrieved
fromcrime scenes, in the determination
ofpaternity , and in the characterization
ofgenetic diversity or breeding patterns in
animal populations.