19.1: Restriction Fragment Length Polymorphism (RFLP)

• Restriction Fragment Length Polymorphism — the first historical method used in forensic DNA testing.

• It utilizes restriction endonucleases that recognize and cleave specific sites along the DNA sequence.

• Appropriate restriction endonucleases should be selected so that the genomic DNA is cleaved at sites that flank the VNTR core repeat region.

• Southern transfer and hybridization technique:

  • The DNA is denatured and transferred from the gel to a supporting matrix such as a nylon or nitrocellulose membrane.

  • The DNA immobilized on the membrane is then hybridized with a labeled probe.

  • Only bands of DNA that have complementary sequences to the probe are recognized by detection systems such as autoradiography.

Common VNTR Loci

Restriction Endonuclease Digestion

• Restriction endonucleases: These are enzymes that cleave the phosphodiester bond of DNA at or near specific recognition nucleotide sequences known as restriction sites.

• A restriction site usually is a short motif that is 4–8 bp in length.

• It often has a specific palindromic recognition sequence, that is, a segment of double-stranded DNA in which the nucleotide’s sequence is identical to an inverted sequence in the complementary strand.

Southern Transfer

• It is also known as Southern blotting.

• This method can be used to transfer DNA from an agarose gel to a solid matrix so that it can be detected with a hybridization probe.

• This technique was named after Sir Edwin Southern.

Hybridization with Probes

• Multilocus Probe Technique (MLP): It can detect multiple VNTR loci simultaneously.

  • It was pioneered by Sir Alec Jeffreys in 1984 and later called DNA Fingerprinting.

• Single-Locus Probe Technique (SLP): It generates a simple pattern called a DNA profile, consisting of one band for a homozygote and two bands for a heterozygote per locus.

Detection

• To detect VNTR loci, a labeled SLP probe is hybridized to the target sequence of DNA, which has been immobilized on a solid matrix such as a piece of nylon membrane transfer.

• Bin: A range of DNA fragments that differ by only a few repeat units.

Factors Affecting RFLP Results

• DNA Degradation

• Restriction Digestion–Related Artifacts

  • Partial Restriction Digestion

  • Star Activity: A deviation of the specificity of a cleavage site of a restriction endonuclease.

  • Point Mutations: Caused by the substitution, deletion, or insertion of a single nucleotide.

• Electrophoresis and Blotting Artifacts

  • Partial Stripping

  • Separation Resolution Limits and Band Shifting

  • Bands Running off Gel

19.2: Amplified Fragment Length Polymorphism (AFLP)

• Amplified Fragment Length Polymorphism (AFLP): A PCR-based technique that uses selective amplification of a subset of digested DNA fragments to generate and compare unique fingerprints for genomes of interest.

• D1S80: Locus used by forensic DNA laboratories for AFLP analysis.

• D1S80 loci are detected as discrete alleles and thus can be compared directly to an allelic ladder on the same gel.

• The AFLP technique requires less DNA than the RFLP method and performs better for degraded samples. The AFLP method at the D1S80 locus can be analyzed in a multiplex fashion with an amelogenin locus.

• The amelogenin gene is used for forensic sex-typing applications. Typing the amelogenin gene enables the determination of the sex of the contributor of a biological sample.