RFLP Application in Mutation Detection

1. RFLP Application in Mutation Detection
By: Saberzadeh
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2. Restriction Fragment Length Polymorphism
(RFLP)
• A technique in which organisms may be differentiated
by analysis of patterns derived from cleavage of their
DNA.
• If two organisms differ in the distance between sites of
cleavage of a particular restriction endonuclease, the
length of the fragments produced will differ when the
DNA is digested with a restriction enzyme.
• The similarity of the patterns generated can be used to
differentiate species (and even strains) from one another.
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3. Applications of RFLP
 can be used in many different settings to accomplish
different objectives:
1- In paternity cases or criminal cases to determine the
source of a DNA sample. (i.e. it has forensic applications).
2- Determining the disease status of an individual. (e.g. it can
be used in the detection of mutations particularly known
muations)
3- To measure recombination rates which can lead to a
genetic map with the distance between RFLP loci
measured in centiMorgans.
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5. Restriction Fragment
Length Polymorphism
Analysis
1. Digest DNA using
restriction enzyme(s)
2. Run digested DNA on gel
using gel electrophoresis
• Smear - Many DNA
fragments with slight
differences in length
1. Expose gel to a chemical to
denature double-stranded
DNA to become single-
stranded
2. Southern blotting

6. RFLP Analysis
4. Southern blotting:
1. Transfer DNA from gel to nylon
membrane
2. Expose nylon membrane to solution
with radioactive complementary
nucleotide probes that hybridize
to specifically chosen DNA
sequences on nylon membrane
3. Place nylon membrane against X-
ray film, where hybridized
radioactive probes cause
exposure of X-ray film,
producing an autoradiogram
http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980211.html

7. RFLP analysis
 Differences in pattern to
detect polymorphisms

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9. Screening of a novel BRCA2 mutation by
rapid in-house PCR-RFLP
(Revista Romană de Medicină de Laborator Vol. 19, Nr. 4/4, Decembrie 2011)
• As BRCA entire gene sequencing is complex, time-
consuming and seriously expensive.
• developing of rapid and cheap pre-screening methods
for targeting each identified mutation offers the
advantage of quick diagnosis,avoiding entire gene
sequencing.
• We imagined a simple and rapid screening for
c.8680C>T mutation in large lots of patients by PCR-
RFLP and agarose gel electrophoresis followed by UV
visualization and interpretation.
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Restriction sites for TaaI on a BRCA2 281bp amplicon of exon 21, for the
wild-type allele (top) or c.8680C>T mutant allele (bottom). Numbers
correspond to the length of products in base pairs (bp).

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