Lab 6 - DNA Fingerprinting Flashcards
(22 cards)
What is another term for DNA fingerprinting?
DNA profiling
What is the purpose of DNA Fingerprinting?
A forensic technique used to identify an individual/sample by their DNA profile (for parentage testing, genealogy, & criminal investigations)
What are the types of polymorphic markers used to differentiate sequences?
RFLPs (Restriction Fragment Lenght Polymorphisms)
VNTRs (Variable Number Tandem Repeats)/ STRs (Short Tandem Repeats)
What are the steps involved in DNA fingerprinting?
- Take a tissue/blood sample from the suspect
- Extract DNA from the sample
- Add restriction enzymes
- PCR amplification
- Separation (Gel electrophoresis or capillary electrophoresis)
- Analysis/Comparison
What are restriction enzymes?
Endonucleases that target specific regions of 4-8bp, catalyze cleavage of phosphate bonds, require Mg2+ ions (co-factor), palindromic sequences, generate 5’ phosphate & 3’ hydroxyl group at cleave site
Also, used as a defence mechanism in bacteria to target foreign viral DNA
What do DNA fragments generated by restriction enzymes depend on?
Distance between recognition sites
How many restriction enzymes are generally required for generation of a fragment of interest? (e.g., cloning)
Two
Name the different causes of differences in recognition sites between individuals
- Different patterns of restriction fragment recognition sites
- Different alleles have differences in their constituent base-pairs (Differences in distribution/frequency of recognition sites)
- Mutations (Creation/Deletion of recognition sites)
- Variations in distance between rec. sites in DNA often caused by intervening repetitive base sequences
What does VNTR stand for?
Variable Number Tandem Repeats
How long are VNTRs?
10-100 base-pair DNA sequences
Where are VNTRs found?
On autosomal & sex chromosomes
What are traits of VNTRs?
- 10-100 base-pair DNA sequences long
- Repeated several times
- Flanked by known sequences that are conserved (and thus targetable)
- Found on autosomal & sex chromosomes
What does STR stand for?
Short Tandem Repeats
How long are STRs?
2-7 base-pair DNA sequences (most commonly 4bp)
What are traits of STRs?
- 2-7 base-pair DNA sequences long
- Repeated several times (variable)
- Flanked by known sequences that are conserved (and thus targetable)
- 1000s of STR locations throughout the genome (can examine multiple loci simultaneously)
- Found on autosomal & sex chromosomes
When performing PCR of STRs, what is the effect of using primers?
Amplifying regions containing repeats
(T/F) When performing PCR of STRs, you cannot add restriction enzymes to the PCR mix.
False
When performing PCR of STRs, what is the effect of including restriction sites on the 5’ end of the primer? Additionally, what is the main use case of this?
To generate compatible ends
Mainly used when cloning
(T/F) Capillary Electrophoresis is slower and less easily automated than gel electrophoresis
False
How do DNA samples that go through capillary electrophoresis appear?
As an electropherogram (graph showing the quantity of light at specific wavelengths detected over time)
When performing capillary electrophoresis, what do DNA fragments appear as and why?
Different-coloured peaks
So they can be easily compared across samples and to the DNA ladder
