DNA analysis and manipulation

Question 1

What are some reasons why we need to manipulate DNA ?

Answer 1

• To help produce recombinant protein (or RNA!) for biochemical / structural analysis
• To sequence DNA (e.g. mutant analysis)
• To complement mutants – linking genotype to phenotype
• To be able to construct specific mutations to investigate gene/enzyme function
• To make fusions to reporter genes – e.g. GFP
• To generate new biochemical pathways and regulatory circuits in bacteria, plants and animals

Question 2

What are functions of plasmid vectors ?

Answer 2

• General DNA cloning and manipulation (e.g. pBluescript II)
• DNA sequencing
• Protein over-expression e.g. pET21
• Gene expression – complementation (organism-specific)
• Gene expression – reporter genes (organism-specific)

Question 3

Explain pBluescript II ?

Answer 3

• A modern cloning vector
  1. Ampicillin resistance gene
  2. pUC origin of replication
  3. Lac operon promoter
  4. Multiple cloning site
  5. lacZα fragment gene
  6. Phage f1 origin of replication (rarely used)

Question 4

Name some examples of restriction enzymes ?

Answer 4

1. AluI
2. EcoRI
3. KpnI

Question 5

Gene amplified by PCR using ?

Answer 5

Chromosomal DNA or cDNA

Question 6

What is the PCR product cloned into ?

Answer 6

• Standard cloning vector and sequenced (to check for mutations)
• Gene moved to different vectors – e.g. for over-expression

Question 7

Extent of DNA is defined by ?

Answer 7

PCR primers

Question 8

What do PCR primers not need to do ?

Answer 8

PCR primers do not need to anneal to the

template DNA at the 5’ end

Question 9

Engineering genes for easy protein purification?

Answer 9

• Mix cut vector and insert
• Add DNA ligase
• Transform E. coli

Question 10

As long as there is sufficient complementarity at the 3’ end. What will an oligo do ?

Answer 10

An oligo can be
extended > 40 nt upstream to incorporate restriction site, ribosome binding site
etc.

Question 11

What do restriction enzymes often not do?

Answer 11

Restriction enzymes often do not cut close to DNA ends, therefore DNA is often blunt-end cloned first, then subcloned

Question 12

What is isolated plasmid usually a mixture of ?

Answer 12

Covalently closed circular (CCC) and open circular (OC)

Question 13

Restriction enzymes cut?

Answer 13

One strand of DNA first (i.e. make OC)

Question 14

What do Partial digests give ?

Answer 14

Greater than expected bands

Question 15

Explain Inverse PCR mutagenesis ?

Answer 15

Once a gene is cloned you can make a mutant allele (e.g. deletion or single amino acid) then reintroduce this into the wild-type strain by homologous recombination to generate a mutant. The mutant protein can also be overexpressed and studied

Question 16

Inverse PCR mutagenesis uses circular template and divergent primers, used for ?

Answer 16

• Amino acid substitutions
• Large deletions
• Small insertions

Question 17

Who invented Gibson assembly ?

Answer 17

Invented by Daniel Gibson at Craig Venter Institute

Question 18

What does Gibson assembly allow to happen ?

Answer 18

Allows simultaneous assembly of several DNA fragments with overlapping (~20 bp) ends

Question 19

What does the Gibson assembly not require ?

Answer 19

• Restriction sites
• No scars are left (seamless)
• Specialised plasmid vectors (unlike Golden Gate cloning)

Question 20

What kind of reaction and process is Gibson assembly ?

Answer 20

• One tube reaction

- Isothermal (PCR not involved in the actual assembly)

Question 21

What is needed for Gibson Assembly ?

Answer 21

Add (at 50°C):

• T5 exonuclease
• Phusion polymerase
• Taq ligase

Question 22

What is the aim of Gibson assembly ?

Answer 22

You aim to assemble 3 DNA fragments each containing a gene or part of a gene into a plasmid in one step

Question 23

In Gibson Assembly your fragments will usually be either ?

Answer 23

• In Gibson Assembly your fragments will usually be either PCR products or
  synthetic DNA fragments (synthesised using oligonucleotides by a company)
• This means the ends of the DNA fragments can be customised

Question 24

PCR products can be produced with ?

Answer 24

Long 5’ tails (e.g. 30 nt tail)

Question 25

Do the tails overlap ?

Answer 25

The tails can overlap between adjacent PCR products by 20-40nt

Question 26

How are Vectors amplified ?

Answer 26

Vectors can also be amplified by PCR or linearised – if linearised the overlap needs to be entirely derived from the outer fragments to be cloned