25. Constructing cDNA libraries

Question 1

what are two sources of DNA for DNA cloning when the DNA sequence is not known?

Answer 1

genomic libraries and cDNA libraries

Question 2

define cDNA

Answer 2

A set of DNA clones that represent the mRNAs present in a given cell at a given time

Question 3

how does cDNA libraries work? 5 steps

Answer 3

1. transcription occurs and mRNA is isolated
2. reverse trancriptase makes a complimentary cDNA strand attached to mRNA
3. use RNase H to make cuts on the mRNA strand
4. DNA polymerase elongates these cuts, filling in with DNA
5. terminal transferase adds cytosines on to the 3’ OH group to help with ligation

Question 4

what does terminal transferace do?

Answer 4

It is a template-independent DNA polymerase. catalyzing the repetitive addition of deoxyribonucleotides to the 3’-OH of oligodeoxyribonucleotides and single- stranded and double-stranded DNA .

Question 5

why is the selection source of mRNA important for cDNA libraries? three points

Answer 5

1. not all cells express the same genes
2. different genes are expressed at different times
3. to obtain mRNA, the gene must be activley expressed

Question 6

what does reverse transcriptase do?

Answer 6

it copies RNA into DNA (this is the opposite way round!)