Biotechnology

Question 1

Steps in PCR

Answer 1

Denaturation
- heating to seperate strands - 96 C
Annealing
- cool so primers can attatch - 55-65 C
Extension
- warm so Taq polymerase extends DNA from primer - 72 C
Repeat 25-35x –> doubles DNA every time

Question 2

Reverse Transcriptase

Answer 2

• makes a copy of DNA from mRNA –> reverse transcription

retroviruses
* RT used to make DNA from RNA template to integrate with host DNA

RT-PCR
* research virsuses
* diagnosis of genetic diseases
* quantatively determine gene expression
* create gene without introns - insertion into prokaryotes

Question 3

Gel Electrophoresis

Answer 3

• occurs after PCR to analyse DNA
• seperates DNA fragments based on molecular size
• DNA molecules (negatively charged) are pushed towards a positive terminal
• smaller the molecule, the faster and further they go
• DNA profiling
• determining species/evoluntionary relationships
• paterntity testing
• forensic science
• identifying fragments created by restriction enzymes

Question 4

CRISPR

Answer 4

• gene editing technique which targets a specific gene
• short sequences of DNA viral origin
• repeated sequences of nucleotides interspaced with old viral DNA

Question 5

Cas proteins (Cas9) and spacers

Answer 5

• Cas9
  defence mechanism of bacteria and archaea
  attatch viral DNA found in bacteria –> cut it up
  inserts copy of old viral DNA into genome (Spacers)

Question 6

DNA hybridisation

Answer 6

• a technqieue that seperates the double stranded DNA of two species into single strands
• these strands are mixed and allowed to bind –> then reheaded and the temperature of seperation recorded
• the higher the temperaure required to seperate the hybrid DNA, the more closely related the species

Question 7

DNA Profiling

Paternity Testing/Forensic Science

Answer 7

Does not require specific sequencing of a DNA sample
Two key steps:
1. PCR with restriction enzyme amplify multiple highly variable STR regions located in the introns
- different primers used for each STR
- DNA fragments are tagged with a fluorescent dye
2. Gel electrophoresis is used to seperate the DNA fragments
- a unique DNA profile is produced for each individual

Paternity Cases –> multiple band matches required
Forensic Cases –> 100% band match required