Polymerase Chain Reaction

Question 1

What fo you need for DNA synthesis in vitro?

Answer 1

• DNA polymerase
• dNTPs
• Template DNA
• Primer

Question 2

What does PCR stand for?

Answer 2

Polymerase chain reaction

Question 3

What fo we need for PCR?

Answer 3

• Templete DNA
• Primers
• dNTPs
• Buffer
• Tag polymerase
• Heat

Question 4

How discovered PCR?

Answer 4

Mullis 1993

Question 5

How many cycles in the process of PCR?

Answer 5

30-40 cycles

Question 6

What are the steps in PCR?

Answer 6

• Denaturation
• Annealing
• Extension

Question 7

Describe the denaturing step in PCR:

Answer 7

• Double DNA stran melts open

- Heating sample to 95C

Question 8

Describe the annealing step in PCR:

Answer 8

-Primers bind to DNA and polymerase attaches and starts copying DNA

Question 9

Describe the extension step in PCR:

Answer 9

72C optimum temperature for polymerase and extension of fragments

Question 10

What is PCR preformed in?

Answer 10

Thermocyler

Question 11

What happens each time the PCR is cycles?

Answer 11

More strands

Question 12

What is the application of PCR described as?

Answer 12

Exponential

Question 13

Which part of the DNA is amplified in PCR?

Answer 13

Between primers

Question 14

Can specific sequences be amplified from complex mixture of DNA?

Answer 14

Yes

Question 15

What are the ends of the amplified fragments defined by?

Answer 15

2 primers

Question 16

What are PCR primers?

Answer 16

short -20 base pairs single stranded DNA (oligonucleotides)

Question 17

Are PCR primers synthesis by naturally or commercially?

Answer 17

Commercially

Question 18

How much DNA do you need to have to visualise on an agarose gel?

Answer 18

1 microgram

Question 19

How do we analyse DNA?

Answer 19

With agarose gel electrophoresis

Question 20

Describe the separation of DNA by size:

Answer 20

• Potential difference applied along gel
• DNA moves to positive electrode through gel depending on conformation (shape)/size (smaller fragments faster than large)
• Stain DNA with fluorescent dye for detection by UV exposure

Question 21

What is the processes called where PCR products being directly sequences?

Answer 21

Sanger Sequencing

Question 22

What are the application of PCR?

Answer 22

• DNA sequencing
• Detection of pathogens in water
• Genetic fingerprinting
• Forensic analysis
• Diagnosis of genetic disorders
• Prenatal diagnosis
• Analysis of ancient DNA

Question 23

What are the limitations of PCR?

Answer 23

• Sequence information is required to design 2 primers
• Limit on length of amplified fragment
• Potentially high error rate
• Very sensitive to exact reaction conditions
• any contaminating DNA will be amplified

Question 24

Do bacteria have plasmids?

Answer 24

Yes

Question 25

What is the shape of bacterial chromosome?

Answer 25

Circular

Question 26

What are plasmids?

Answer 26

small extrachromosomal circles of DNA

Question 27

What can DNA be cut by?

Answer 27

Restriction endonucleases

Question 28

What can join DNA?

Answer 28

DNA ligase forms phosphodiester bond (requires ATP)

Question 29

What is recombinant DNA?

Answer 29

Allows two DNA molecules to be joined

Question 30

What is good about able being able to join DNA?

Answer 30

Genes can be inserted into plasmids

Question 31

What are the overlap-based methods of DNA assembly?

Answer 31

PCR-based methods - Site-specific recombination methods - DNA repair-based methods - In vivo homologous recombination

Question 32

Describe the gene cloning:

Answer 32

- Introduce recombinant plasmid into bacterial cell - Replicated - Cell divides - Clone of cells - Recover DNA for analysis - Gene cloning

Question 33

What is another way you can clone genes?

Answer 33

Inserting gene into bacteriophage DNA vectors

Question 34

What is a gene library?

Answer 34

- Collection of DNA put into plasmids and multiplied through transformation in a bacteria - Collection of Recombinant clones

Question 35

What is DNA hybridisation?

Answer 35

screening for clones containing gene of interest

Question 36

What is meant by transgenic?

Answer 36

- Genes between species | - Generic code is universal

Question 37

How can DNA be expressed in cells?

Answer 37

- Introduction of DNA into cell - Expression vector inserted withe gene of DNA - Can express gene in bacteria by inserting into a plasmid

Question 38

What is in an expression vector?

Answer 38

a particular type of plasmid that has a promoter telling a bacteria to transcribe the gene

Question 39

What has to happen before DNA can be expressed in cells?

Answer 39

Due to introns in mRNA is needed | -Reverse transcribed into cDNA before cloned into vector

Question 40

What are the applications of genetic engineering?

Answer 40

- Human insulin - Blood clotting factor VIII - Human growth hormone - Bovine chymosin - Hepatitis B vaccine - Artemisinin

Question 41

What is SRY gene?

Answer 41

Sex determination gene

Question 42

Give an example of genetic engineering?

Answer 42

- SRY genes make xx mice phenotypically male | - Inserting XY into XX cause male characteristics

Question 43

What is a reporter gene?

Answer 43

Proteins tagged with green fluorescent protein

Question 44

What is synthetic biology?

Answer 44

design of new biological parts, device, and systems and re-design existing, natural biological systems for useful purpose

Question 45

What are the applications of synthetic biology?

Answer 45

-Artemisinin production (didn't have to wait for crop production which effect the economical price)