R.O Lecture 2

Question 1

Give examples of cloning whole genetic material

Answer 1

• Of an individual being (whole genomic material), e.g. Dolly the Sheep
• Of a cell, e.g. E.coli (genetic material copied and passed on
  to progeny when cells replicate)

Question 2

Give an example of partial genetic material

Answer 2

• molecular cloning
  -Of a DNA sequence, e.g. one gene or part of a gene, by PCR
  (polymerase chain reaction) or by transforming into a cell for replication

Question 3

Give a quick description of the process of recombinant DNA technology

Answer 3

-Identify genetic material of interest (DNA of interest)
-Produce copies of ‘DNA of interest’
-Insert ‘DNA of interest’ into a vector (e.g. plasmid)
-Introduce vector (containing ‘DNA of interest’) into cell (expression system)

Question 4

Brief history of Polymerase chain reaction (PCR)

Answer 4

-Kary Mullis (1983 87)
-Investigating sickle cell anemia (SNP) but sample was limiting
-PCR: replication of a specific segment of DNA
-1992: Hoffmann La Roche purchased licence from Cetus.
-1993: Nobel prize in Chemistry

Question 5

What was Mullis’s invention (PCR) based on?

Answer 5

Based on two principles:
Natural DNA replication in cells
Sanger Sequencing Method

Question 6

Describe the process of Natural DNA replication in cells

Answer 6

-Base pairing enables existing DNA strands to serve as templates for new complementary strands (A:T and G:C)

-Allows replication of genetic information, which is to be passed on to daughter cells during cell division.

-The enzyme DNA polymerase synthesises complementary DNA strand (needs nucleotides, Mg 2+ ions, other enzymes to separate DNA strands).

-Synthesis starts from complementary primer (Primase makes
complementary RNA primer)

-Replication always proceeds in 5’ to 3’ direction

Question 7

DNA Polymerase

Answer 7

The enzyme DNA polymerase synthesises complementary DNA
strand (needs nucleotides, Mg 2+ ions, other enzymes to separate DNA strands).

It can only bind and start adding on from the 3’ bend

Question 8

Where does synthesis begin? (natural dna rep in cells)

Answer 8

Starts from the complementary primer.

Question 9

What makes the complementary RNA primer

Answer 9

Primase

Question 10

Describe Sanger Sequencing Method

Answer 10

*invented by Sanger, Smith and Coulson in the mid 1970s
*based on synthesis of a complementary DNA strand by DNA polymerase (from E.coli )
*initiated by a short oligonucleotide primer

(DNA replication in a tube)

Question 11

Function of primers

Answer 11

*Mullis introduced a second ‘reverse’ primer
*primers FRAME your target sequence (requires prior knowledge of the sequence)

Question 12

Benefit of PCR

Answer 12

Allows us to make billions of copies of a specific DNA sequence

Question 13

What does the PCR amplification process involve?

Answer 13

Replication process involves thermal cycling

Question 14

Name the processes in PCR Amplification

Answer 14

1. Denaturation
2. Annealing
3. Extension/Elongation

Question 15

Denaturation

Answer 15

94 °C
- Heat briefly to separate DNA strands

Question 16

Annealing

Answer 16

52-65 °C
- Cool to allow primers to form hydrogen bonds with complementary DNA sequence
- Annealing temperature (Ta) picked based on melting temperature (Tm) of primers

Question 17

How is the Annealing Temperature determined

Answer 17

Ta~ 5 °C below lowest Tm of primers
Annealing temperature depends on the length and composition of the primers used.

Question 18

Extention/Elongation

Answer 18

72 °C
*DNA polymerase binds and using primers, extends new sequence
*Each cycle lasts 1- 5 minutes and at the end the targeted DNA sequence
is doubled
*The solution is then heated again starting the next cycle of denaturing, primer binding and DNA synthesis

Question 19

What is the number of cyles for the thermal cycle

Answer 19

30

Question 20

Why was Taq polymerase incorporated instead of E.Coli DNA polymerase?

Answer 20

• Changed from using E. Coli DNA polymerase to heat stable Taq polymerase (resistant to denaturation)
• Temperature cycling now performed in single instrument

Question 21

Name the components for PCR reaction

Answer 21

*PCR Buffer
*dNTPs
*DNA Template
*Forward Primer
*Reverse Primer
*Taq polymerase
*dH20

Question 22

What type of PCR Buffer is used?

Answer 22

PCR Buffer specific for the polymerase but is usually
Tris based pH buffer (pH 8 8.8;
*most now contain MgCl 2 )
Polymerase is usually added last (immediately before reaction starts)
activity declines the longer it is above 0 C

Question 23

Describe Taq DNA Polymerase

Answer 23

• Integral to ‘cycling’ of PCR: Need a polymerase that is not denatured during high temperature steps
  *Produced by T hermus aq uaticus , a bacteria found in hot springs/hydrothermal vents (thermophile)
  *heat stable DNA polymerase; elongates DNA at 72 C; resistant to denaturation step of PCR
  *Isolated in 1976, Science ‘Molecule of the Year’ 1989

Question 24

Name other heat-stable polymerases

Answer 24

Pfu and Vent, isolated from other thermophiles both of these have proof-reading capacity

Question 25

Describe the proof reading ability of Pfu

Answer 25

*Pfu DNA polymerase (from Pyrococcus furiosus) works its way along the DNA from the 5’ end to the 3’ end (of new strand) and corrects nucleotide mis-incorporation errors.

*Pfu error rate is 1 every 7.7 × 105 to 1 × 106 bp (6x better than Taq)

Question 26

What type of genetic material can be used for cloning?

Answer 26

cDNA or gDNA can be used as a template for PCR

Question 27

cDNA

Answer 27

generated as complementary DNA from mature mRNA (no introns), mRNA is an indicator for gene expression (rather than just the presence of a gene)

Question 28

Exon

Answer 28

Coding portion of DNA

Question 29

Introns

Answer 29

non-coding portion of DNA that take up a huge amount of genetic code.

Question 30

What is the appropriate template for recombinant protein production

Answer 30

The template should be cDNA to ensure introns have been removed

Question 31

Uses of Genomic DNA (gDNA)

Answer 31

*Clinical (genetic screening or diagnosis, identification of pathogens (virus, bacteria, fungi),
tissue typing)
*Forensics (use of DNA in criminal/missing persons cases, paternity tests)
* Evolutionary/Historic studies (Identify using DNA from bones, teeth, Phylogenetics)
*Research (sequencing studies, site-directed mutagenesis)

Question 32

It is important that primers…

Answer 32

*Be complementary to 3’ end of the target sequence (at least 15 bp or more)

*Have a melting temperature (T m ) in the range 52 to 72 C, with no more than 5 C difference within a pair

*Have a GC content between 40 60 % ideally

*Not bind to other areas within the DNA sequence

*Not bind to each other (inter or intra primer pairing)

*should not form excessive secondary structures (hairpins), 3’ end has to be free for elongation

Question 33

What can occur if primer concentration is too high?

Answer 33

Can lead to primer dimer formation (of primers have complementary regions) and non-specific priming.

Question 34

dNTP concentration

Answer 34

*Nucleotides required to build new copies of DNA (A, T, G, C)
*Requires enough dNTP to be incorporated during 30-40 cycles, Taq
polymerase is more accurate at low concentrations of dNTPs

Question 35

Mg2+ concentration

Answer 35

*DNA polymerase only works in the presence of free Mg 2+ ions (co factor)
*Concentration has an effect on the annealing temperature and the
specificity of the reaction.

Question 36

DMSO

Answer 36

*Reduces secondary structure that could inhibit the progress of the
polymerase (used at 5 10 %)

Question 37

What do you do with your PCR product?

Answer 37

*Run it on a gel to confirm the size of the product
*Sequence it directly: sanger or NextGen sequencing
*inset it into a vector