USE OF ALLELIC VARIANTS IN DNA FINGERPRINTING

Introduction

Dr. Danielle Bond presents a comprehensive overview of DNA fingerprinting, including its methodology, applications, and the implications surrounding its application in criminal forensics and paternity testing.

Learning Objectives

  • Describe short tandem repeats (STRs) and their role in DNA fingerprinting.
  • Explain the steps involved in performing DNA fingerprinting.
  • Discuss the various applications of DNA fingerprinting, particularly in parentage testing and criminal cases.

Case Study: Murder on Campus

  • The scenario describes a murder in the Great Hall involving Barry Parker, President of the Newcastle Knife and Sword Club (NKSC), who was found dead after a heated argument during the club's AGM.
  • Three suspects were present during the argument, and DNA evidence collected from under the victim's fingernails raises the need for DNA fingerprinting to identify the murderer.

DNA Fingerprinting Overview

  • Human DNA Similarity: 99.9% of human DNA is identical among individuals.
  • Variable Regions in DNA: DNA fingerprinting focuses on variable regions to identify specific individuals or familial relations.
  • Tandem Repeats: Sequences in which two or more nucleotides are repeated adjacently, which are significant in DNA profiling.
  • Variable Number Tandem Repeats (VNTRs): Specifically, short tandem repeats (STRs) are utilized for DNA fingerprinting due to their variability in repeat numbers across alleles and individuals. Typically, a standard profile involves 13 different loci for accurate comparison.

Methodologies for DNA Fingerprinting

Restriction Fragment Length Polymorphism (RFLP)
  • Principle: RFLP assays measure the length of VNTRs.
  • Process:
    1. Use restriction enzymes to cleave DNA at specific sites outside VNTRs.
    2. Apply electrophoresis to separate the resulting DNA fragments by size.
    3. Visualize length differences to identify allelic variants.
DNA Fingerprinting by RFLP

A detailed process involving:

  1. DNA Cleavage and Electrophoresis: DNA is cleaved, and the resulting fragments are separated in a gel based on size.
  2. Blotting: DNA is transferred onto a nitrocellulose filter.
  3. Probe Hybridization: Filters are exposed to a radioactive probe to identify specific DNA sequences.
  4. Film Development: Filters are developed on photographic film to visualize the hybridized bands. This creates specific patterns or bands on the film based on the location of DNA matches.
Key Questions in RFLP
  • What determines the action of a restriction enzyme on DNA?
  • Identifying the type of blotting performed (Southern Blot) based on the context.
  • The relevance of the probes used in the process and other methods (e.g. PCR) that could focus on specific DNA loci.
DNA Fingerprinting by PCR
  • Quantitative fluorescent polymerase chain reaction (QF-PCR): Amplifies loci of interest using:
    • Sequence-specific primers with fluorescent tags.
    • Capillary gel electrophoresis is utilized to separate PCR amplicons based on size differences.
  • A peak analysis may indicate the number of alleles present at a particular locus, with single or multiple peaks denoting different genetic inheritances.

Interpretation of Results

  • Probe patterns for each suspect's DNA will provide insights into their potential involvement in the crime based on matches with the DNA evidence obtained from the victim.

Case Conclusions: The Murderer

  • Analysis of the DNA markers allows for the identification of the murderer from the suspects based on match comparisons at the STR loci.

Paternity / Parentage Testing

  • Overview of using DNA fingerprinting to determine biological relations, particularly in cases contesting inheritance claims.
  • Each child inherits one allele from the mother and one from the father at each STR locus, providing a framework for comparison.
  • Procedures involve obtaining samples from both parents and children, with STR loci analyzed to identify allele matches confirming paternity.

Linking and Haplotype Analysis

  • Haplotype Definition: Refers to a set of linked alleles inherited together from a single parent.
  • Uses in Genetic Analysis: Important for investigating genetic traits, particularly when specific mutations remain unidentified.
  • Linkage Analysis: Viable for tracking genetic mutations associated with diseases, utilizing STR markers to ascertain inheritance patterns within families affected by disorders such as Angerus murderus (an autosomal dominant disorder).

Summary

  • DNA fingerprinting can compare allelic variants efficiently using RFLP or QF-PCR, applicable in various contexts, including paternity testing and forensic investigations. Each child must have one allele from each parent at every locus for parentage tests, and all alleles from a suspect must match the evidence at all loci in forensic cases.

Recommended Reading

  • Principles of Genetics, Snustad and Simmons, 7th ed. is suggested for continued study on DNA fingerprinting and genetic principles.