Recombinant DNA

Question 1

What is the basic strategy of Recombinant DNA ?

Answer 1

1. Extract DNA from mould
2. Break DNA into fragments
3. Ligate into cloning vector
4. Create library in E. coli
5. Identify correct gene
6. Transfer to expression/shuttle vector
7. Express in cat

Question 2

What are Cloning Vectors ?

Answer 2

• These are autonomously replicating pieces of DNA that allow a molecular biologist to amplify and manipulate another piece of DNA that they are interested in
• Although in the post-PCR era it is possible to manipulate DNA without replicating it in a host cell most molecular biology experiments will still involve the cloning of DNA into a vector at some stage

Question 3

Once in a cloning vector the DNA will ?

Answer 3

• Normally then be introduced into E. coli. This bacterium has been developed as an ideal host for a range of cloned DNAs
• Note that other cloning hosts are available such as yeast and insect/mammalian cells

Question 4

Fragmenting Genomic DNA

Answer 4

Physical: Shearing/ Sonication
Enzymatic: Non-random

Question 5

Explaining Introducing Plasmids into E.coli ?

Answer 5

• Treat cells with certain chemicals (eg CaCl2) to increase permeability
• Mix cells with DNA and pass an electric current through mixture (electroporation)
• Spread cells on an agar plate containing appropriate antibiotic

Question 6

The bigger the genome the more ?

Answer 6

Individual clones you need to ensure that you have the gene that you are after. For genomes with a lot of non-coding DNA then most clones will be useless

Question 7

cDNA synthesis first strand synthesis ?

Answer 7

cDNA is commonly generated from mRNA for gene expression analyses such as RT-qPCR and RNA-seq. mRNA is selectively reverse transcribed using oligo-dT primers that are the reverse complement of the poly-adenylated tail on the 3’ end of all mRNA

Question 8

cDNA synthesis second strand synthesis ?

Answer 8

The one-tube system allows second strand cDNA to be synthesised immediately after the first strand reaction, thereby improving the yield of double-stranded cDNA by eliminating extraction and precipitation between steps. Furthermore, the double-stranded cDNA can be cloned into either plasmid or bacteriophage vectors

Question 9

Southern Blotting technique?

Answer 9

1. Digest the DNA
2. Run the digest on an agarose gel
3. Denature the DNA
4. Transfer the denatured DNA to the membrane
5. Probe the membrane
6. Visualise your radioactively labeled target sequence

Question 10

What probes can you use for Southern Blotting ?

Answer 10

• Probes can be RNA, ssDNA, denatured dsDNA or oligonucleotides
• They need to be labelled with a marker which could be radioactivity, a fluorophore, or a ligand (to which an antibody for example could bind)

Question 11

What is an expression vector with example ?

Answer 11

• The expression vector is a plasmid engineered to introduce a particular gene into the target cell.
• An example of expression vector is the plasmid used to produce insulin important for treating diseases such as diabetes

Question 12

What is a shuttle vector ?

Answer 12

A plasmid that has both bacterial and eukaryotic origins of replication and so can propagate in either kind of cell; useful as a form of recombinant DNA for growth in a bacterium and subsequent transfer into eukaryotic cells for expression

Question 13

How retroviral vectors are used in gene therapy?

Answer 13

The most important advantage that retroviral vectors offer is their ability to transform their single stranded RNA genome into a double stranded DNA molecule that stably integrates into the target cell genome. This means that retroviral vectors can be used to permanently modify the host cell nuclear genome.