Genetic Engineering 1

Question 1

Clone

Definition

Answer 1

a group of genetically identical organisms

e.g. a bacterial colony

Question 2

DNA Cloning

Definition

Answer 2

• the propagation of a specific DNA fragment isolated from its genomic context
• the chemical purification of a specific DNA sequence

Question 3

Genetic Engineering Entails…

Answer 3

… the isolation and purification of specific genes (gene cloning)
… determination of the DNA sequence of these genes
… alteration of these genes in the test tube
… reintroduction of the engineered gene into a suitable host organism

Question 4

Genetic Engineers Toolkit

Answer 4

Restriction Enzyme - cuts DNA reproducibly
DNA Ligase - joins DNA molecules together
Vector - autonomously replicating DNA molecules
Host - organism in which the vector will replicate

Question 5

Restriction and Modification of Bacteriophage Growth

Experiment

Answer 5

1) bacteriophage λ, virulent to E.coli stain C, infects strain C cells producing a high titre of phage plaques
2) phage particles isolated from the infected strain C cell plaques and used to infect E.coli strain K cells
3) only a few plaques from on the strain K plate, growth of the bacteriophage is restricted in strain K
4) phage particles isolated from strain K plaques
5) strain K cells reinfected with the isolated phage particles
6) this time a high titre of plaques is produced (same as original infection of strain C)
7) phages have become modified to allow phage growth in strain K

Question 6

Restriction and Modification of Bacteriophage Growth

Explaination

Answer 6

• bacterial cells have mechanisms to protect them from phage infection
• DNA entering the bacterial cell that is recognised as foreign is cut into fragments by a bacterial endonuclease, a restriction enzyme
• in order to protect its own DNA the sites recognised by the endonuclease in the bacterial genome are modified by methylation
• in strain C bacteriophage DNA is not recognised as foreign so a high titre of plaques is able to form
• after initial infection of strain K, in most cells the endonuclease is able to fragment the phage genome, but in the few that this doesn’t happen, phage DNA is modified by the bacterial cell before the endonuclease can digest it and is then protected from the enzyme
• all of the phages that are able to form plaques have had ther DNA modified so when they are isolated and used to reinfect strain K there DNA is already protected and a high titre of plaques is produced

Question 7

Agarose Gel Electrophoresis

Answer 7

• DNA is placed in wells at the top of the gel
• an electric current is passed through the buffer solution covering the gel
• the negatively charged DNA migrates along the gel towards the positive electrode
• the speed it migrates at is relative to the length of the molecule
• large fragments migrate slowly
• small fragments move faster
• distance travelled is inversely proportional to the logarithm of the fragment size in base pairs

Question 8

Detecting DNA on Agarose Gel

Answer 8

• staining with ethidium bromide which intercalates and binds to DNA between base pairs
• it glows pink under UV light

Question 9

How to determine the size of a DNA fragment using an agarose gel?

Answer 9

• use a marker, a sample containing fragments of known size
• plot a graph of distance migrated (x) against length of fragment (y) using a logarithmic scale for the fragments in the marker
• draw a line of best fit
• measure the distance migrated by the fragments in the sample and read off the graph want length this corresponds to

Question 10

Restriction Enzymes

Type I

Answer 10

• recognise a specific sequence in DNA and bind to it
• migrate an indeterminate number of bases and starts excising nucleotides (from 1 strand)
• does not give rise to a specific fragment pattern

Question 11

Restriction Enzymes

Type II

Answer 11

• recognises a specific DNA sequence

- cuts precisely and reproducibly at that site

Question 12

Restriction Enyzme Recognition

Sites

Answer 12

-palindromic

Question 13

Restriction Enzymes

Cuts

Answer 13

• some restriction enzymes make staggered cuts leaving sticky ends
• some restriction enzymes make flush cuts leaving blunt ends

Question 14

Length of Restriction Sites of Restriction Enzymes

Answer 14

• recognition sites can be 4, 6, 8 etc. base pairs long
• the longer sequence, the less likely that that sequence will occur in DNA so it is possible to predict the frequency at which the enzyme will cut a particular piece of DNA
• an n base pair restriction enzyme will cut DNA on average every 4^n bases
• this means that it is possible to generate different size fragments using different restriction enzymes

Question 15

Mapping Genomes With Restriction Enzymes

Answer 15

• it is possible to map where the restriction sites are for different restriction enzymes by digesting a DNA sample with different combinations of restriction enzymes
• run the digested sample on an agarose gel

Question 16

Southern Blotting

Answer 16

1) run gel and stain to check migration of markers
2) soak gel in 0.4M NaOH to denature DNA in situ
3) set up blot
- tank containing transfer solution
- bridge and wick
- gel placed on top
- positively charged nylon transfer membrane placed over gel
- stack of filter paper towels placed on top
- glass plate and weight on top
4) the negatively charged DNA moves through the transfer solution and is attracted to the nylon
5) the filter carries a replica of the agarose gel in the form of single stranded DNA
6) the filter can be hybridised with a radioactively labelled clone sequence which will anneal to complimentary sequences by base pairing

Question 17

How to radio label DNA

Answer 17

1) denature the double helix by heating to produce single stranded DNA
2) anneal with random sequence hexanucleotides (siz nucleotide fragments of DNA)
3) add DNA polymerase and 32-p-dNTPs
4) DNA copied will be short fragments that are radio\
actively labelled

Question 18

Autoradiography

Answer 18

• start with a hybridised southern blot
• when the filter is exposed to x-ray film the position of the hybridised fragments is revealed by blackening of the film
• the radioactive probe can be thought of as a gene specific stain
• it can be used to detect fragments derived from very large genomes

Question 19

What can southern blotting be used for?

Answer 19

-can be used to detect different alleles of individual genes in individual members of a population

Question 20

DNA Fingerprinting - CF Example

Answer 20

• the recessive disease allele is associated with another mutation which causes the loss of the HindIII site outside of the CF gene coding region
• take DNA sample and digest with HindIII
• run on an agarose gel
• tag with probe
• the presence of 5kb fragments means that the DNA has been cut so the healthy CF allele is present
• the presence of 7kb fragments means that the DNA has not been cut so the mutation is present in the HindIII binding site so the disease allele is present

Question 21

Good Cloning Vectors

Answer 21

…are small - few sites for restriction enzymes other than those used for cloning DNA fragments
…contain genetic markers for selection - to enable easy selection of transformants and recombinants
…reproduce avidly in host - produce large quantities of DNA

Question 22

DNA Ligase

Answer 22

• joins DNA molecules

- makes a phosphodiester bond between terminal 5’ phosphate on one DNA molecule and the terminal 3’ -OH on another

Question 23

Cloning Vector

Definition

Answer 23

-an autonomously replicating DNA molecule that reproduces avidly within its host

Question 24

Inserting Genomic DNA into Plasmid

Answer 24

1) start with plasmid e.g. pBluescript, 2.9kb, with an ampicillin resistance gene not usually found in bacteria, multiple cloning site within beta galactosidase gene
2) digest plasmid with hindIII, plasmid splits at the cloning site in the beta galactosidase gene
3) immediately adjacent to the cloning site are 2 sequences to which universal primers can anneal
4) digest vector with alkaline phosphate, it removes the phosphate group from the 5’ end to minimise self ligation
5) mix genomic digest with vector and DNA ligase
6) mix with E.coli cells
7) select for ampicillin resistant cells that appear white as they contain recombinant plasmids

Question 25

Possible Ligation Products

Answer 25

i) self ligated genomic DNA ii) self ligated plasmid iii) recombinant plasmid DNA - growing with ampicillin will destroy cells that haven't taken up a plasmid - beta galctosidase turns colourless galactose (X-Gal) into an insoluble blue dye - in cells with recombinant plasmids the beta galactosidase gene is disrupted - add X-Gal to the cells that could survive the ampicillin, cells that appear white contain recombinant plasmids

Question 26

Do we still need to construct cloned libraries?

Answer 26

- originally only cloned DNA could be sequenced - modern DNA technologies mean that it is not always necessary - but for assembly of accurate genome sequences are still helpful to have large insert clones - it is still necessary to obtain clones of individual genes or cDNAs for experimental applications

Question 27

Shotgun Cloning

Answer 27

1) cut up genome at random 2) ligate into vector 3) transform into host 4) host replicates producing many copies of the DNA

Question 28

Two Landmark Technologies in Genetic Analysis

Answer 28

i) the polymerase chain reaction | ii) high through-put whole genome sequencing

Question 29

DNA Sequencing

Answer 29

- essential tool in modern genetics - reveals coding capacity of genes - permits precise genetic manipulation - permits comparative analysis of individuals in a population and between species

Question 30

Dideoxynucleotides

Answer 30

-lack a 3' -OH so one it has been added no further nucleotides can be added to the molecule

Question 31

Extending a DNA / RNA molecule

Answer 31

- grow when nucleotides are added to their 3' ends | - phosphodiester bond forms between 5' phosphate of incoming dNTP and 3' -OH of the molecule

Question 32

Sequencing By Synthesis - Automated Sequencing

Answer 32

1) denature DNA strand to form single strands 2) anneal a primer to form a template for DNA polymerase 3) add DNA polymerase, deoxyNTPs (normal bases) and dideoxyNTPs (terminator bases) 4) the newly synthesised strand will end when a dideoxyNTP is added 5) as there are so many molecules, you will end up with fragments of all lengths each ending in a dideoxyNTP, each dideoxyNTP is also fluorescently tagged with a different tag for each base, G, C, A and T 6) run on a polyacrylamide gel 7) at the end of the gel a laser excites the fluorescent label so that a detector can read it 8) fragments are allowed to run off the gel, so the fragements are read from smallest to largest by the detector 9) this gives a sequence of fluorescent labels corresponding to the base sequence of the DNA