Module 3 - Recombinant DNA Technology

Question 1

What is gene cloning, and which techniques are used to accomplish it?

Answer 1

Gene cloning is the isolation and amplification of a given gene.
Restriction enzymes (scissors), DNA ligase (glue) and plasmids (DNA production lines) are all components of gene cloning and recombination.

Question 2

Define blunt & sticky ends.

Answer 2

Blunt ends are fromed when dsDNA is cut by restriction enzymes in the middle of a sequence.
Sticky ends are formed when staggered rather than straight cuts are made.

Question 3

What are isoshizomers?

Answer 3

Restriction enzymes that recognise the same sequence but makes different cuts.

Question 4

Describe the function of DNA ligase.

Answer 4

Repairs phosphodiester bond by catalysing formation of covalent bonds between each DNA strand.

Question 5

What are the two methods of transformation (inserting recombinant DNA back into bacteria)?

Answer 5

1. Heat shock, for chemically competent cells

2. Electroporation, for electro-competent cells

Question 6

What are the three essential features of Cloning Vectors?

Answer 6

1. Origin of replication
2. Dominant selectable marker
3. At least one unique RE cleavage site.

Question 7

In ascending order, what are the four vectors alternative to plasmids?

Answer 7

1. Bacteriophage - 23kb inserts
2. Cosmid vectors - 50kb
3. Bacterial artificial chromosomes (BAC’s) - 150-300kb
4. Yeast artificial chromosomes (YAC’s) - 200-500kb

Question 8

Four essential features of Expression Vectors are?

Answer 8

Promoter, operator, termination sequence and ribosomal binding site.

Question 9

Explain the function and components of a shuttle vector.

Answer 9

Shuttle vectors allow for replication in both species - prokaryotes (bacteria) as well as eukaryotes. They require an origin (species 1 & 2), selectable marker (sp. 1 & 2), appropriate marker (sp. 2) & ribosomal binding site (sp. 2)

Question 10

Explain the mechanism by which can DNA be amplified.

Answer 10

A Polymerase Chain Reaction (PCR):

1. denatures dsDNA using heat (92 - 95’C)
2. anneals primers to ssDNA through cooling (40 - 65’C)
3. extends DNA via DNA polymerase (72’C)
4. repetition (amplified product is called the amplicon)

Requires template DNA containing GOI, nucleotides (AGTC), primers (ss, free 3’OH), >15bps, DNA pol, Mg ions & salts, water.

Question 11

What are the four limitations of PCR?

Answer 11

1. knowledge of DNA sequence required
2. contamination
3. accuracy of Taq polymerase
4. size of amplicons is limited

*Taq pol lacks proofreading ability, more errors present.

Question 12

How does ligation occur?

Answer 12

1. Isloate the DNA by digestion with restriction enzymes.
2. Combine 2 DNA sources and incubate under annealing conditions.
3. Treat annealed DNA fragments with DNA ligase.

Question 13

What are the steps involved in cloning?

Answer 13

1. Cut plasmid vector and DNA fragment to be cloned with the same RE(s).
2. Add insert to plasmid and ligate.
3. Transform recombinant DNA into bacteria by heat shock or electroporation.
4. Select cells that contain recombinant DNA.
5. Grow the cell.

Question 14

What are the two major methods of screening transformed bacteria?

Answer 14

Antibiotic sensitivity, colour screening.

Question 15

There are two main types of DNA libraries. Define each.

Answer 15

Genomic libraries - a genomic DNA library is a set of DNA clones that collectively contain the entire genome of any given organism.
cDNA libraries - a combination of cloned cDNA fragments inserted into a collection of host cells, which together constitute a portion of the transcriptome of the organism (only transcribed DNA).

Question 16

What is Taq polymerase?

Answer 16

Thermus aquaticus - a thermostable polymerase that attaches nucleotides to DNA templates at high temperatures.

Question 17

A probe is a short (up to ___ bp) ___________ made up of ____ or ____ and is complementary to a sequence within the _____ ___ _______. Probes can be labelled, either with _______ or ________ ____.

Answer 17

50, oligonucleotide, DNA, RNA, gene of interest. Radiation, fluorescent dye.

Question 18

Southern Blotting locates a gene from a mixed population of DNA, and its probe can be DNA or RNA. What are the key steps in Southern Blotting?

Answer 18

1. Digestion - DNA of interest is digested by REs to generate smaller fragments.
2. Separation - desired DNA is separated by argarose gel electrophoresis.
3. Transfer - separated DNA fragments are transferred onto a solid support nylon membrane.
4. Denaturation - the transfer of DNA is done under denaturing conditions.
5. Labelling - hybridised with labelled DNA probe.

Question 19

NoRtheRn Blotting locates _____ that corresponds to the gene of interest. It is _____ based and its probe can either be _____ or _____. It occurs via the physical process of ______ ______, the same as the Southern Blot.

Answer 19

mRNA, RNA, RNA, DNA. Capillary action.

Question 20

Describe the Western Blot.

Answer 20

The Western Blot occurs with proteins and a probe of antibodies. This technique separates proteins using gel electrophoresis SDS-PAGE. SDS is an anionic detergent, proteins are transferred from a gel into a nitrocellulose membrane. Individual proteins are detected using primary and secondary antibodies. Occurs via electricity.

Question 21

Define a bacteriophage.

Answer 21

Bacteriophage are viruses that infect bacteria, inserting their DNA into the cell in order to utilise the cell machinery to make more phage.

Question 22

Explain the protection of DNA by methylation.

Answer 22

Bacteriophage injects viral DNA at the recognition site. The same recognition site on the bacterial DNA is methylated (methyl group added) by EcoR1 methylase. RE’s are able to degrade the viral DNA at the recognition site, but is unable to reach the bacterial DNA in order to cleave it due to the presence of a methyl group.

Question 23

The two methods of screening DNA libraries is by _______ _______ (expressive vector -> protein product) or by _______ _______ (radioactive probes).

Answer 23

Direct selection, molecular hybridisation.

Question 24

Describe the Sanger-Dideoxy method of DNA screening.

Answer 24

Sanger-Dideoxy or terminator chemistry can be map based or shotgun. Each ddNTP reaction is performed in a different tube with P-labels to allow detection. It is performed on an acrylamide gel to give better resolution that argarose gel. This sequence produces complimentary strands at 5’-3’.

Question 25

Describe the Automated approach to DNA sequencing.

Answer 25

Where Sanger is manual, each ddNTP has a differently coloured fluorescent tag attached. All four ddNTP reactions are performed in the same tube. A fluorescent detector analyses the position of each tag as they pass through the gel and the software gives a point out.

Question 26

Describe the map based and shotgun approaches to DNA sequencing.

Answer 26

Map based - chromosome is partially digested with RE’s and ligated into YACs or BACs to create libraries (contigs). Large insert clones are aligned using genetic marker or RE maps, regions of overlapping fragments are then broken up and “sub-cloned”. Map before sequencing, more difficult.
Shotgun - fragments are inserted into plasmids and cloned in bacteria. Overlapping fragments sequenced using automated systems. Scattered approach. Sequencing is random before assembly. Faster, easier.

Question 27

What are some applications of PCR?

Answer 27

• DNA profiling (amplifying Short Tandem Repeats)
• Mutagenesis (oligonucleotide directed)
• Amplifying genes or DNA of interest
• RT qPCR (measuring transcripts for high/low expression)
• Melt curves (used for detecting species of nucleotide sequences and SNPs)