March 27

Question 1

How is DNA library assembled using Truseq?

Answer 1

1. DNA isolation
2. DNA fragmentation, size selection
3. Blunt ends and phosphorylate them
4. Add poly A tails to 3’ ends
5. Addaptor ligation
6. PCR amplification
7. Sequencing and library quantification

Question 2

Following double strand cleave with RE, how are these ends blunted? (2)

Answer 2

Polymerase has 5’3’ activity to fill in to match a 5’ overhang, and the 3’-5’ exonuclease activity of polymerase will chewback the 3’ overhang

Question 3

What is used to phosphorylate 5’ ends of DNA for sequencing that normally only have 5’ Oh group/

Answer 3

T4 polynuclotide kinase

Question 4

DNA pol 1 has what polymerase adn exonuclease activities?

Answer 4

It has 5’-3’ polymerase and exonuclease activities, and also 3’-5’ exonuclese activity

Question 5

What is a Klenow fragment? What about exo-Klenow? What is exo-Klenow used for?

Answer 5

It is large fragment produced when DNA pol 1 is mutated. This fragment is only a part of the full polymerase, it loses the 5’-3’ exonuclease activity

Exo-Klenow means the pol 1 also does not have 3’-5’ exonuclease activity. It is used to add non-template poly A tail to 3’ end of DNA molecules

Question 6

Once we add 5’ PO4 and 3’ Poly A tail to teh dsDNA, what needed on Y-adaptors that we add next? What done next?

Answer 6

The Y adaptors are 2 single strands of DNA that are base paired for an end, form a Y. The joined part of the adaptor has 1 strand with poly T overhang to complement 3’ Poly A overhang, and the other strand has a 5’ PO4 so it can join onto the poly A tail

Then do PCR

Question 7

What are transposomes?

Answer 7

It is when form protein that has part of DNA from donor DNA, then this protein containing DNA strand inserts itelf into new strand

Question 8

What is solid phase reverse immobilization?

Answer 8

It is when DNA and RNA in impure sample, the impurities will bind to beads. The beads are magnetic and so can remove beads with magnet.