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DNA Fingerprinting RFLP Guide

RFLP, or restriction fragment length polymorphism, is a DNA fingerprinting technique used to distinguish between individuals of the same species using DNA samples. It involves breaking down DNA into fragments using restriction enzymes, separating the fragments by size through gel electrophoresis, and analyzing the pattern of fragment distribution to identify related DNA samples. A standard curve is created to determine the size of DNA fragments based on how far they have moved through the gel during electrophoresis.

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142 views15 pages

DNA Fingerprinting RFLP Guide

RFLP, or restriction fragment length polymorphism, is a DNA fingerprinting technique used to distinguish between individuals of the same species using DNA samples. It involves breaking down DNA into fragments using restriction enzymes, separating the fragments by size through gel electrophoresis, and analyzing the pattern of fragment distribution to identify related DNA samples. A standard curve is created to determine the size of DNA fragments based on how far they have moved through the gel during electrophoresis.

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ikhsas yogia
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Restriction Fragment Length

Polymorphism (RFLP)

Dept. Biochemistry & Molecular


Biology FMUI
DNA Fingerprinting/RFLP
• A technique used by scientists to distinguish
between individuals of the same species using
only samples of their DNA

• It is also termed DNA profiling


Stages of DNA Fingerprinting
• Stage 1:
Cells are broken down
to release DNA

If only a small amount of DNA


is available it can be
amplified using the
polymerase chain reaction
(PCR)
Stages of DNA Fingerprinting
• Step 2:
The DNA is cut into fragments using restriction enzymes.

Each restriction enzyme cuts DNA at a specific base sequence.


Restriction site
Enzyme Site
Palindrome
Recognition

• Each enzyme digests


(cuts) DNA at a
specific sequence =
restriction site
• Enzymes recognize
4- or 6- base pair,
palindromic
sequences
(eg GAATTC)

Fragment 1 Fragment 2
Stages of DNA Fingerprinting
Stage 3:
• Fragments are
separated on the basis
of size using a process
called gel
electrophoresis.
• DNA fragments are
injected into wells and
an electric current is
applied along the gel.
Stages of DNA Fingerprinting
DNA is negatively charged
so it is attracted to the
positive end of the gel.
The shorter DNA
fragments move faster
than the longer
fragments.
DNA is separated on basis
of size.
Stages of DNA Fingerprinting
Stage 4:
• The pattern of fragment distribution is then
analysed.
DNA Fingerprinting Procedures
Analysis of Stained Gel

Determine
restriction fragment
sizes

• Create standard
curve using DNA
marker

• Measure distance
traveled by
restriction fragments

• Determine size of
DNA fragments

Identify the related


samples
Fingerprinting Standard Curve: Semi-log

Molecular Weight
100,000

Determination

Size (bp) Distance (mm) 10,000

Size, base pairs


23,000 11.0 B

9,400 13.0
1,000
6,500 15.0

4,400 18.0

2,300 23.0
100
A
2,000 24.0 0 5 10 15 20 25 30
Distance, mm

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