28 Jun DEMONSTRATION OF PATERNITY TEST USING DNA FINGERPRINTING
Experiment goal:
The goal of this experiment is to develop a basic understanding of DNA fingerprinting. Variations in
restriction enzyme cleavage patterns (cuts) obtained from different DNA samples (child, mother, and
potential fathers) will be analyzed and the biological father of a child will be determined using DNA
fingerprinting.
Background:
DNA fingerprinting involves the separation by electrophoretic analysis of either, DNA fragments cut
by restriction enzymes, or tandem repeats regions amplified by the polymerase chain reaction (PCR).
Individuals are then identified on the basis of their respective DNA profile. Restriction enzymes are
endonucleases that cut double-stranded DNA. The points of cut occur in or near very specific sequences
of bases called recognition sites. Generated fragments vary in size according to differences in restriction
enzyme cleavage patterns. Tandem repeats are segments of DNA that contain sequences from 2 to 40
bases in length repeated in tandem manner many times (e.g. AGAGAGAG….). The number of segments
or “core units” repeats varies among individuals of the same species. PCR
Polymorphic DNA refers to chromosomal regions that vary widely from individual to individual.
By examining several of these regions within the genomic DNA obtained from an individual, one may
obtain a “DNA fingerprint” for that individual. The most commonly used polymorphisms are those which
vary in length; these are known as Restriction Fragment Length Polymorphisms (RFLPs) and
Variable Number of Tandem Repeats (VNTRs).
Overview of the experiment:
In this experiment, DNAs are digested by restriction enzymes and the fragmentation patterns serve as the
individual fingerprint. The DNA fragmentation patterns can be analyzed directly in the stained agarose
gel. In this hypothetical case, DNA obtained from two potential fathers (Tubes D&E) and mother (B) and
child (C) are cleaved with one restriction enzyme in separate reactions. The objective is to analyze and
match the DNA fragmentation patterns after agarose gel electrophoresis and determine if Individual 1 or
Individual 2 is the Father of the Child.
Steps involved in DNA Fingerprinting:
1- Extraction of high molecular weight, duplex DNA from blood, semen, skin, on hair roots, and
purification.
2- PCR amplification to increase the number of DNA copies for the analysis.
3- Restriction enzyme digests to produce fragments of various sizes (RFLPs).
4- Agarose gel electrophoresis to separate the fragments according to size.
5- Visualization of fragments using methylene blue stain.
6- Comparison of fragment sizes of potential fathers with those from child and mother.
1. Why do different individuals such as siblings have different restriction
enzyme recognition sites?
2. What is the function of PCR primers used in DNA paternity analysis?
3. Why is there more than one single locus used in an actual paternity DNA
test?
4. Why do we not use probes in this DNA paternity simulation and still
obtain results?
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