TOPIC 6: Forensics

Question 1

What are Short Tandem Repeats (STR)?

Answer 1

• short repeated sequences (3-7 bases) found in non-coding DNA
• involved in chromatic folding and transcription regulation

Question 2

How do STRs create a unique DNA profile?

Answer 2

• number of STRs at a particular locus can vary on each chromosome
• each individual has a large number of STR loci

Question 3

Summarise the process of DNA profiling

Answer 3

1. Obtain DNA sample
2. Create fragments
3. Separate fragments
4. Visualise fragments

Question 4

How are DNA samples obtained?

Answer 4

• cheek cells from mouth swabbing

- WBCs obtained in blood sample

Question 5

How are DNA fragments created?

Answer 5

restriction enzymes (endonuclease) cut DNA at specific recognition sites

Question 6

Why is the use of restriction enzymes no longer necessary for isolating STR fragments?

Answer 6

specific primers can be made to isolate each STR

Question 7

What are DNA primers and why are they needed?

Answer 7

short DNA sequences complementary to either side of the STR

starting point for DNA synthesis by taq polymerase

Question 8

List the steps of the PCR cycle

Answer 8

denaturation, annealing, extension

Question 9

What is Taq polymerase?

Answer 9

thermostable DNA polymerase

Question 10

Why are dNTPs used for extension in PCR?

Answer 10

DNA polymerase catalysed formation of phosphodiester bond between nucleotides and diphosphate is released

Question 11

What happens during denaturation in PCR?

Answer 11

H bonds break and DNA strands are separated

Question 12

What temperature is need for DNA denaturation?

Answer 12

95°C

Question 13

What is PCR used for?

Answer 13

amplification of DNA sequences, creating longer repeating sequences

Question 14

What happens during annealing in PCR?

Answer 14

primers attach at the end and start of STR sequences via complementary base pairing.

Question 15

At what temperature does annealing take place and why?

Answer 15

55°C

for specific binding of primer

Question 16

What happens during extension in PCR?

Answer 16

Taq polymerase synthesises complementary DNA strands using free nucleotides (dNTPS)

Question 17

What is gel electrophoresis?

Answer 17

a method of separating DNA fragments according to size using agarose gel by applying an electric field

Question 18

Describe the steps for gel electrophoresis

Answer 18

• 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

Question 19

Why do DNA fragments move from - to +?

Answer 19

negatively charged phosphate group attracted to +be charge of anode

Question 20

Why do small fragments migrate further?

Answer 20

they can move past gel particles

Question 21

How can you visualise DNA banding pattern by staining?

Answer 21

ethidium bromide inserts itself between base pairs

glows in UV light

Question 22

What is the difference between staining DNA directly and Southern blotting?

Answer 22

Southern blotting allows SPECIFIC DNA to be stained

Question 23

Describe the steps of Southern blotting

Answer 23

• 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