TOPIC 6: Forensics Flashcards
What are Short Tandem Repeats (STR)?
- short repeated sequences (3-7 bases) found in non-coding DNA
- involved in chromatic folding and transcription regulation
How do STRs create a unique DNA profile?
- number of STRs at a particular locus can vary on each chromosome
- each individual has a large number of STR loci
Summarise the process of DNA profiling
- Obtain DNA sample
- Create fragments
- Separate fragments
- Visualise fragments
How are DNA samples obtained?
- cheek cells from mouth swabbing
- WBCs obtained in blood sample
How are DNA fragments created?
restriction enzymes (endonuclease) cut DNA at specific recognition sites
Why is the use of restriction enzymes no longer necessary for isolating STR fragments?
specific primers can be made to isolate each STR
What are DNA primers and why are they needed?
short DNA sequences complementary to either side of the STR
starting point for DNA synthesis by taq polymerase
List the steps of the PCR cycle
denaturation, annealing, extension
What is Taq polymerase?
thermostable DNA polymerase
Why are dNTPs used for extension in PCR?
DNA polymerase catalysed formation of phosphodiester bond between nucleotides and diphosphate is released
What happens during denaturation in PCR?
H bonds break and DNA strands are separated
What temperature is need for DNA denaturation?
95°C
What is PCR used for?
amplification of DNA sequences, creating longer repeating sequences
What happens during annealing in PCR?
primers attach at the end and start of STR sequences via complementary base pairing.
At what temperature does annealing take place and why?
55°C
for specific binding of primer
What happens during extension in PCR?
Taq polymerase synthesises complementary DNA strands using free nucleotides (dNTPS)
What is gel electrophoresis?
a method of separating DNA fragments according to size using agarose gel by applying an electric field
Describe the steps for gel electrophoresis
- mix agarose and buffer & microwave to melt agarose
- cool and pour into mould
- add comb and remove when gel sets
- gel put into tank & DNA samples loaded into wells with a pipette
- DNA fragments move towards the anode
Why do DNA fragments move from - to +?
negatively charged phosphate group attracted to +be charge of anode
Why do small fragments migrate further?
they can move past gel particles
How can you visualise DNA banding pattern by staining?
ethidium bromide inserts itself between base pairs
glows in UV light
What is the difference between staining DNA directly and Southern blotting?
Southern blotting allows SPECIFIC DNA to be stained
Describe the steps of Southern blotting
- DNA gel electrophoresis
- DNA transferred to nylon or nitrocellulose membrane
- membrane with bound DNA removed
- hybridisation: membrane incubated with fluorescent DNA probe which binds with target DNA via comp. base pairing
- DNA probe visualised
