PCR Cloning Engineering

Question 1

Why is Mg2+ buffer required?

Answer 1

To help DNA polymerase to work.

Question 1

What is the polymerase chain reaction? What is required for it?

Answer 1

Amplification of DNA:
- template DNA
- primers
- dNTPs
- buffer (Mg2+)
- Taq polymerase
- thermocycler

Question 2

Where is the name Taq polymerase derived from?

Answer 2

1968 - Thomas Brock discovered microbes in hot spring, Yellowstone National Park.
Name is derived from bacteria Thermus aquaticus.

Question 3

Why can’t standard DNA polymerase be used in PCR?

Answer 3

DNA strands are separated by heat to break hydrogen bonds which would denature standard DNA polymerase.

Question 4

What did Kary Mullis win the Nobel Prize for in 1993?

Answer 4

Invention of PCR method.

Question 5

Give the process of PCR.

Answer 5

1. Denaturation = heated to 94 degrees Celsius to separate strands.
2. Annealing = cooled to 50-65 degrees Celsius so primers can bind to DNA and polymerase can attach and copy DNA.
3. Extension/DNA synthesis = At 72 degrees Celsius (optimum temperature for polymerase) to allow extension of fragment.

Question 6

What is the relationship between double-stranded DNA molecules and cycles?

Answer 6

Exponential.

Question 7

How many DNA molecules will there be at the end of the first cycle of PCR?

Answer 7

2

Question 8

How many DNA molecules will there be at the end of the second cycle of PCR?

Answer 8

4

Question 9

How many DNA molecules will there be at the end of the thirtieth cycle of PCR?

Answer 9

1073741824

Question 10

Which sequence of DNA is amplified in PCR?

Answer 10

The region between the primers.

Question 11

What does PCR allow to be amplified from a complex mixture of DNA?

Answer 11

Specific sequences.

Question 12

How are the ends of the amplified fragment defined?

Answer 12

By the 2 primers.

Question 13

What are primers?

Answer 13

Oligonucleotides = 20 base pair single stranded DNA.

Question 14

How are primers synthesised?

Answer 14

Chemically.

Question 15

What are the uses of PCR?

Answer 15

Any research uses in which a specific piece of DNA needs to be amplified:

• detection of pathogens in water
• DNA sequencing
• diagnosis of genetic disorders (if gene and
  primers are known)
• prenatal diagnosis
• analysis of ancient DNA
• genetic fingerprinting (court cases for
  paternal issues)
• forensic analysis

Question 16

What are the limitations of PCR?

Answer 16

• sequence information is required to
  design 2 primers
• limit on length of amplified fragment
• potentially high error rate (e.g. Taq
  polymerase ~ 10^-4)
• very sensitive to exact reaction conditions
  so not easily quantified
• tiny amounts of contaminating DNA also
  amplified

Question 17

What is agarose gel electrophoresis?

Answer 17

Polysaccharide powder dissolved in buffer to form gel to separate DNA.

Question 18

What is the purpose of loading dye?

Answer 18

To help track how far DNA sample has travelled and allows sample to sink into gel.

Question 19

What happens when an electric current is applied to gel?

Answer 19

DNA moves through gel to positive electrode because it it negatively charged.

Question 20

How does conformation shape affect the speed at which DNA travels through the gel?

Answer 20

Fastest = supercoiled plasmids as they cause less friction.

Slower = linear.

Slowest = circular.

Question 21

How do smaller fragments move through the gel compared to large ones?

Answer 21

Faster.

Question 22

Why is DNA stained with ethidium bromide?

Answer 22

Fluorescent dye for detection by UV exposure.

Question 23

What is a DNA ladder used for?

Answer 23

Standardised way of measuring what sizes DNA samples are.

Question 24

What is required for Sanger sequencing?

Sanger sequencing, developed by Frederick Sanger in 1977, is a method used to determine the exact sequence of nucleotides (A, T, C, G) in a segment of DNA.

Answer 24

DNA polymerase.
Deoxyribonucleotides modified to dideoxy ribonucleosides.
Template DNA.
Primer.

Question 25

What is the role of dideoxy ribonucleosides?

Answer 25

Terminates chain so DNA doesn’t elongate further because it has hydroxyl group at 3’ carbon missing.

Question 26

Describe the process of sanger sequencing.

Answer 26

1. Primer added to single strand DNA
   fragment to be sequenced.
2. Add excess amounts of unlabelled dNTPs
   and small amounts of labelled chain-
   terminating ddNTPs.
3. Products = mixture of DNA each
   containing a chain-terminating ddNTP
   labelled as a specific fluorescent marker.
4. Loaded onto capillary gel (allows
   separation of macromolecules/nucleic
   acids whose mass to charge ratios don’t
   vary much in size).
5. Each band from gel electrophoresis is
   read by a computer and size-separated
   products are read in sequence.
6. Each peak represents a single nucleotide.

Question 27

Why is Sanger sequencing useful?

Answer 27

To sequence nucleotides of amplified DNA for answering biological questions.

Question 28

Explain how DNA is stored in bacteria.

Answer 28

Bacterial genomes are typically a circular chromosome.

Bacteria often harbour plasmids (small extrachromosomal circles of DNA).

Question 29

How is DNA cut when manipulating genes?

Answer 29

By restriction endonucleases (enzymes extracted from bacteria).

Question 30

How do restriction endonucleases work?

Answer 30

They scan and cleave at specific palindromic sequences (read both ways) to leave straight/sticky ends.

Question 31

What is the process called when a combination of restriction endonucleases and DNA ligase is used?

Answer 31

Genetic engineering.

Question 32

Where is the cleavage site of HaeIII?

Answer 32

5’ GG/CC 3’
3’ CC/GG 5’

Question 33

Where is the cleavage site of EcoRI?

Answer 33

5’ G/AATTC 3’
3’ CTTAA/G 5’

Question 34

Where is the cleavage site of HindIII?

Answer 34

5’ A/AGCTT 3’
3’ TTCGA/A 5’

Question 35

What is the equation for working out how many times a specific endonuclease will cut the genome?

Answer 35

Sites = length of genome (bp) / length of palindromic sequence (bp)

Question 36

What is the role of DNA ligase?

Answer 36

Allows 2 DNA molecules to be joined to form recombinant DNA.

Forms phosphodiester bonds (covalent linkage).

Requires ATP.

Question 37

What is one way you can form recombinant DNA?

Answer 37

Insert DNA fragment to be cloned into bacterial plasmid.

Question 38

Give the process of gene cloning.

Answer 38

1. Introduce recombinant plasmid into bacterial cell. The gene is replicated when the cell divides.
2. Clone cells and recover DNA for analysis.
3. Many copies of the purified recombinant plasmid are isolated from lysed bacterial cells.

Question 39

Give an alternative processes to clone genes.

Answer 39

• Cloning genes by insertion into
  bacteriophage vectors.
• DNA inserted into plasmids when bacteria
  are infected in laboratory
  for amplification/to make a library.

Library = a collection of genetic material fragments stored and propagated/transmitted in population of microbes through process of molecular cloning (to produce exact gene copies).

Question 40

What is transgenics?

Answer 40

Genes between species. Taking a gene from one organism and expressing it in another.

Question 41

Why does transgenics work?

Answer 41

The genetic code is universal (minus a few exceptions).

Question 42

An example of transgenics is producing human insulin.

Explain how this happens.

Answer 42

Human insulin gene inserted into E.coli.

Large-scale fermentation.

Insulin purified and harvested to give commercial insulin.