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RFLP Technique in Genetic Analysis

RFLP (Restriction Fragment Length Polymorphism) is a molecular marker used to detect differences in DNA sequences through enzyme digestion and gel electrophoresis, primarily for genetic testing of disease-causing mutations. The document discusses the process of RFLP analysis, including the use of CAPS (Cleaved Amplified Polymorphic Sequence) to amplify and screen DNA fragments for polymorphisms. Applications of RFLP include forensics, mutation detection, and genetic mapping.

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15 views11 pages

RFLP Technique in Genetic Analysis

RFLP (Restriction Fragment Length Polymorphism) is a molecular marker used to detect differences in DNA sequences through enzyme digestion and gel electrophoresis, primarily for genetic testing of disease-causing mutations. The document discusses the process of RFLP analysis, including the use of CAPS (Cleaved Amplified Polymorphic Sequence) to amplify and screen DNA fragments for polymorphisms. Applications of RFLP include forensics, mutation detection, and genetic mapping.

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sainivetha97
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© All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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RFLP-PCR

PHARMACOGENOMICS PRESENTATION
by S SAI NIVETHA
M TECH BIOTECHNOLOGY
RFLP-Restriction Fragment Length
Polymorphism
• Restriction fragment length polymorphisms, or RFLPs, are
differences in homologous DNA sequences that can be detected
by the presence of fragments of different lengths cut by
enzymes.

• RFLP is a molecular marker which is specific to a single


clone/restriction enzyme combination.

• It is mostly used in genetic testing to find mutant


disease-causing gene.
Restriction
digest

Gel
electrophoresis

THE OVERALL
Denaturatio
PROCESS n

Blottin
g

Hybridization and
visualization
PROBES
Single- or low-copy
genomic DNA or cDNA
clones
Cleaved Amplified Polymorphic
Sequence-CAPS
• In CAPS assay we amplify DNA fragments that are digested with a
restriction endonuclease to display RFLP.
CAPS markers

• The RFLP probe is sequenced first


• Primers are designed to amplify 800–2,000-bp DNA fragments.
Targeting introns or 3' untranslated regions should increase the
chance of finding polymorphisms
• The PCR product is cloned and sequenced.
• PCR is performed to amplify DNA fragments from target genotypes,
and the amplicons are separately digested with one or more
restriction enzymes.
• The digested amplicons are screened for polymorphism.
Example - two different alleles for human ß-globin. The normal allele has three restriction enzyme cleavage sites, but
a mutation that causes sickle-cell anaemia also results in an allele that has only two cleavage sites. As shown in the
figure below, the two different alleles display different patterns following gel electrophoresis and Southern
hybridization.
Sickle-cell anaemia - hypothetical pedigree for parents each heterozygous for the sickle-cell allele is shown in the
figure below. The inheritance of the normal and sickle-cell allele in this family can be followed by RFLP-analysis. Each
of the different genotypes, homozygous normal (AA), heterozygous carrier (Aa), and homozygous recessive with
sickle-cell anaemia (aa) can be identified by RFLP analysis.
APPLICATIONS
• Forensics
• Detection of mutations
• Genetic mapping
• CAPS helps in estimating heterozygosity.
• RFLP alleles (morphs) can be used as diagnostic tools.

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