Lecture 25

Question 1

mRNA produces what type of library?

Answer 1

cDNA library

Question 2

A genomic library is made from which molecule?

Answer 2

DNA

Question 3

What is a cDNA library?

Answer 3

Contains all mRNA in an organism (from genes being expressed in that cell).

Question 4

How do you construct a cDNA library?

Answer 4

1. isolate mRNA (oligo - dT column)
2. Anneal oligo - dT primer and copy the DNA using reverse transcriptase.
3. Treat with RNA seH (attacks RNA of RNA/DNA hybrid strands) -> left with only DNA.
4. Add DNA Polymerase I to make dsDNA
5. Clone into plasmid or vector.

Question 5

You can’t amplify with PCR unless….

Answer 5

… The sequence is already known (so you can then design primers).

Question 6

Which library has all genes in an organism?

Answer 6

BAC library.

Question 7

The BAC genomic library includes what in it?

Answer 7

Introns, promoters, repeat sequences, transposable elements, etc.

Question 8

Plasmid cDNA libraries have what?

Answer 8

Coding regions and adjacent transcribed sequences only.

Question 9

Which library has a lower complexity?

Answer 9

cDNA library.

Question 10

How big are the inserts in cDNA?

Answer 10

1-2kb on average (plasmid or phage vectors)

Question 11

How many genes per clone does the BAC genomic library hold?

Answer 11

Multiple (or partial) genes per clone.

Question 12

What method do you use to find a gene with hybridisation?

Answer 12

DNA probe. Use a probe from another species with 70% bp homology (using PCR). Finding a sequence with high Met and Trp is good as there is only one sequence for this. “Degenerate probe”.

Question 13

What method is used to find a gene detecting an expressed protein?

Answer 13

Using antibodies. Need to purify and inject into the animal, then make an expression library.

Question 14

Which method is used to clone conserved core functions?

Answer 14

Complementation of E.coli/yeast mutants.