Jones - Biological Techniques

Question 1

how do phage insert their genomic DNA into a host?

Answer 1

Phage lambda inserts its genomic DNA into a bacterial cell eg E. coli
The complementary cos sites circularise
The Att site on the circular phage lambda DNA attaches to the Att site on the E. coli genome
Recombination of phage lambda and E. coli genome
(looks like a little loop-the-loop on DNA)

Question 2

which enzyme is responsible for joining the cos sites in phage DNA?

Answer 2

DNA ligase

Question 3

describe both steps of ligation, include a brief description of the enzymatic mechanism

Answer 3

1st step (non-enzymatic):
DNA has to collide by chance and stay together long enough for ligase to join them.
This is the most inefficient part and requires a low temperature
2nd step (enzymatic):
Lysine residue in ligase adds an adenylate (AMP) group to the PO4- group
The 3’ OH group attacks the PO4- group and forms a phosphodiester bond
DNA ligase requires ATP as a cofactor

Question 4

what is the optimal temperature for DNA ligation?

Answer 4

16oC is the optimal temperature for this; compromise between enzymatic temperature (25oC) and DNA collision temperature. A higher temperature would mean the collisions would be too brief for ligase to join the phosphodiester bonds

Question 5

which 2 scientists discovered restriction and modification in phage?

Answer 5

Salvadore Luria and Werner Arber

Question 6

define restriction and modification

Answer 6

restriction: the ability for host bacteria to degrade foreign phage DNA, means phage can’t enter new bacterium easily
modification: the ability for phage to enter new bacterium after adopting their methylation pattern

Question 7

briefly describe the differences and uses of the type I and III, and type II restriction endonucleases

Answer 7

Type I and III:
Cleave DNA sites away from recognition sequences
Not used in most molecular biology applications
Type II:
Cleave both DNA strands at specific recognition site
Most abundant form
Widely used in molecular biology

Question 8

describe the nomenclature of restriction enzymes

Answer 8

First 3 letter of name is an abbreviation of the bacteria it was obtained from
4th letter is the strain of bacteria
Number is the type of enzyme used

Question 9

how do restriction enzymes cleave DNA?

Answer 9

Initial binding is loose and doesn’t involve catalytic activity
- If it reaches its recognition site then it binds
- If it doesn’t find its recognition site after 50 bp then it hops/jumps along DNA
Once it’s bound to its recognition site it, and the DNA, undergo conformational changes
The catalytic component becomes activated, and in the presence of Mg2+ the enzyme cleaves both strands by breaking the phosphodiester bonds

Question 10

what is molecular cloning?

Answer 10

Creation of recombinant DNA molecules and their replication in a host organism

Question 11

describe the difference in transformation and transfection

Answer 11

Transformation: recombinant molecule in bacteria
Transfection: recombinant molecule in animal cell

Question 12

describe in brief the steps in molecular cloning

Answer 12

Restriction enzymes produce fragment, can be purified by gel electrophoresis
Fragment ligated into open plasmid
New recombinant molecule is transferred into a cell
Host cell replicates itself – plasmids can be harvested

Question 13

name 3 types of vector

Answer 13

plasmid
phage
cosmid

Question 14

state when and why plasmids, phages and cosmids are used in molecular cloning

Answer 14

Plasmid:
- Naturally occurring multicopy (makes multiple copies)
- =<10kb insert maximum
- subcloning and downstream manipulation, cDNA cloning and expression assays
Phage:
- Bacteriophage λ
- 5-20kb insert maximum
- Genomic DNA cloning, cDNA cloning and expression
Cosmid:
- Plasmid containing a bacteriophage λ cos site (uses the bacteriophage method of gene insertion but no bacteriophage cell lysis)
- 34-45kb insert maximum
- Genomic library construction

Question 15

name 3 essential components of a cloning vector that mean it can be used in molecular cloning

Answer 15

A selectable marker (ie a resistance gene)
Restriction sites to clone the fragment in
Own origin of replication

Question 16

name a significant problem that can occur in molecular cloning, in particular when a fragment is inserted

Answer 16

self-ligation of the vector

Question 17

name 2 ways in which self-ligation is prevented?

Answer 17

1) Treat vector with (alkaline) phosphatase to remove 5’ P groups – phosphodiester bonds can’t form
2) Use of 2 different restriction enzymes – results in the restriction sites combining therefore it can’t be recognised by the 2 restriction enzymes you’ve used. Also means you can be sure of fragment orientation.

Question 18

name a pro and a con of blunt end cloning

Answer 18

pro: don’t need matching sites (if both ends are blunt)
con: 100x less efficient than sticky ends

Question 19

name 2 blunt end restriction enzymes

Answer 19

EcoRV and SmaI

Question 20

what does taq polymerase do?

Answer 20

Taq polymerase extends complementary strands as well as adding a singular adenine base on the 3’ end of the DNA and vector

Question 21

what does a polynucleotide kinase do?

Answer 21

adds phosphate groups to the 5’ end of sequences

Question 22

explain why polynucleotide kinases and phosphatases are used in blunt end cloning

Answer 22

PCR products (without Taq) are missing 5’ P groups as normal primers aren’t phosphorylated – polynucleotide kinase phosphorylates the 5’ ends
Blunt ended vector should be treated with a phosphatase to remove its 5’ phosphate groups and reduce the likelihood of self-ligation

Question 23

how is an overhang converted into a blunt end?

Answer 23

T4 DNA polymerase or the Klenow fragment of DNA polymerase I

• Fills in 5’ overhang in presence of dNTPs
• 3’ –> 5’ exonuclease activity will remove 3’ overhang

Question 24

what is TA cloning?

Answer 24

a type of blunt end cloning
DNA taq polymerase adds a 3’ adenine (A). this means that the vector requires a thymine overhang
the 2 are ligated together

Question 25

how is a T overhang prepared?

Answer 25

Prepare vector by blunt end cutting followed by ddTTP addition using terminal transferase
- Use ddTTP as it results in chain termination – strand doesn’t grow

Question 26

name the 3 methods of bacterial transformation and briefly describe how they work

Answer 26

CaCl2:
- Membranes become leaky by interaction of Ca2+ ions and the -ve membrane
Electroporation:
- Drives DNA into cells by a strong electric current
Heat shock:
- Temperature raised to 42oC – membrane poration and DNA enters

Question 27

how are laboratory bacterial vectors adapted to avoid degradation of foreign, insert DNA?

Answer 27

Lab adapted bacterial strains don’t have restriction enzymes

Question 28

which 3 antibiotics are used for selection and amplification in molecular cloning

Answer 28

ampicillin
tetracycline
kanomycin

Question 29

how do ampicillin, tetracycline and kanomycin stop bacterial growth?

Answer 29

Amp:
- Inhibits bacterial cell wall synthesis by disrupting peptidoglycan cross-linking
- β-lactamase encoded by the resistance gene
Tet:
- Inhibits binding of aminoacyl tRNA to the 30S ribosomal subunit
- Resistance gene product is membrane bound and prevents tetracycline accumulation by an efflux mechanism
Kan:
- Inactivates translation by interfering with ribosome function
- Gene product inactivates kanamycin by phosphorylation

Question 30

in blue-white screening what colour and the recombinant and non recombinant colonies?

Answer 30

Non-recombinant colonies are blue

Recombinant colonies are white

Question 31

how does blue-white screening work?

Answer 31

Plasmid contains lac Z 5’ sequence and the fragment DNA contains the lac Z 3’ sequence. When the recombinant DNA is inserted it disrupts the 5’ sequence of lac Z and therefore it doesn’t encode β-lactamase. As the β-lactamase enzyme is produced when no recombinant molecule is made, this is shown by the breakdown of added metabolite X-gal which is cleaved. When X-gal is cleaved it turns from colourless to blue.

Question 32

name 4 steps that can be carried out after a molecule has been cloned

Answer 32

Further screening of the colony – check if the bacterial sequence is right
Larger scale culture
Plasmid purification – get back pure plasmid from bacteria
Expression analysis – plasmid moved to other cell to see if the protein is still expressed