Restriction Fragment Length Polymorphism

RELEVANT LEARNING OUTCOME:

(a) Describe the principles and procedures of these molecular techniques:

Restriction Fragment Length Polymorphism

🕐Estimated time for this section: 25 minutes

Restriction Fragment Length Polymorphism

Definition: Restriction fragment length polymorphism (abbreviated RFLP) refers to differences (or variations) among people in their DNA sequences at sites recognized by restriction enzymes.

SNP

Single nucleotide polymorphisms (SNPs) can result in the loss of a restriction site.
When digested with the restriction enzyme EcoRI, two fragments are formed from (A).
In contrast, only one fragment is formed from (B) because the polymorphic site has been altered and can no longer be recognised by EcoRI.

VNTR

Varying number of tandem repeats (VNTR): the higher the number of VNTR, the longer the resulting restriction fragments

Application of RFLP

👁️‍🗨️ Watch the video to understand about general idea of application of RFLP;

How is it important in the analysis of Inheritance? (1:38 onwards)

Watch this to prepare for the next segment which is on disease detection using RFLP.

1) Disease detection: Sickle Cell Anaemia

Recall:

Which gene is mutated in patient with sickle cell anaemia?
What do you call this type mutation?
Which amino acid is changed (state the position in the polypeptide chain and the type of amino acid)?

Restriction sites for Ddel restriction enzyme (RE)

Base on the diagram below,

How many restriction sites (for Ddel RE) are there in a normal and mutant beta globin allele?
How many restriction fragments are there after these alleles are digested with Ddel RE?

normal allele: ___________
sickle-cell mutant allele: ____________

RFLP analysis (1st method)

DNA is extracted from individual's body cells
PCR is conducted to amplify beta globin gene
Digestion of DNA with RE
Gel electrophoresis is conducted
Visualize all bands using ethidium bromide. (shown in diagram below)

RFLP analysis (2nd method)

DNA is extracted from individual's body cells
Digestion of DNA with RE (diagram on the top left)

3 restriction sites, 1,2,3 on normal allele, A --> 2 fragments
one missing restriction site, 2 on mutant allele, a --> one large fragment

Gel electrophoresis is conducted
Southern blotting is carried out
Nucleic acid hybridisation using labelled probe (red rectangle) --> only complementary to specific sequence on both allele (one fragment x and fragment y will be identified)

Take note: Every individual will have 2 alleles of beta globin gene:

(Diagram on the left: banding pattern for genotype AA, aa and Aa)

For individual with 2 copies of dominant alleles (homozygous AA)

1 thick band, containing 2 copies of the shorter fragments

For individual with 2 copies of recessive alleles (homozygous aa)

1 thick band, containing 2 copies of the longer fragments

For individual with 1 copy of dominant allele and 1 copy of recessive allele (heterozygous Aa)

1 thin band containing 1 copy of shorter fragment
1 thin band containing 1 copy of longer fragment

Diagram below: The inherited paternal and maternal chromosome of each homologous pair have different number of tandem repeats, and this is unique to each individual. When the genome of individual A and B are separated using gel electrophoresis, unique banding patterns are produced for the individual.

2) DNA Fingerprinting

DNA fingerprinting is a well known method to determine variation among individuals of population at DNA level.

VNTR Analysis

Unique DNA fingerprint for each individual. (diagram on the left)
VNTR are heritable, so the banding patterns of two closely related individuals will be more similar than two unrelated individuals.

Method:

DNA is extracted from individual A, B and C (from the crime scene)
Digestion of DNA into fragments by Restriction enzymes
PCR was done to amplified VNTR from chromosomes 2, 7, and 16
Gel electrophoresis is conducted to separate the different length of VNTR.
Transfer of ssDNA to nitrocellulose membrane (Southern Blotting)
Nucleic acid hybridisation (using probe) and detection is done using autoradiography

🖊️Check Your Understanding: Learning Goals

Attempt Qn 21-22 of the Molecular Techniques Learning Goals

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