mRNA

Question 1

5’ cap of mRNA

Answer 1

7-methylguanosine is joined to the 5’ end of eukaryotic mRNA’s in an unusual 5’,5’-triphosphate linkage

Question 2

5’ RACE

- rapid \_\_\_\_\_\_\_\_\_\_\_
- Anneal \_\_\_\_\_\_\_\_\_\_\_\_
- \_\_\_\_\_\_\_\_\_\_\_\_ & \_\_\_\_\_\_\_\_ with a \_\_\_\_\_\_\_
- \_\_\_\_\_\_\_\_\_\_ w/ adaptors-primers
- cloning into \_\_\_\_\_\_\_\_\_\_
- Most likely problem \_\_\_\_\_\_\_\_\_\_\_

Answer 2

• [rapid] amplification of cDNA ends
  
  • [Anneal] gene-specific primer
  • reverse transcription [&] addition of homopolymeric tail [with a] terminal transferase
  • PCR amplification [w/ adaptors-primers]
  • [cloning into] plasmid vector

Question 3

Drawbacks to 5’ race

Answer 3

• many parital cDNAs are present, so 5’ race product is a mixture of fragments of various lengths, so it can be difficult to get to the 5’ end of full-length DNA

Question 4

Possible improvements to the 5’ race method

Answer 4

• isolate the longest cDNA product before you do homopolymeric tailing
  
  • purify the longest product from a reaction and subject this product to a second round of amplification
  • selection/enrichment of full length cDNAs

Question 5

5’ anchor ligation
is an improved method for _________
the ______ groups from the ___ of _______ mRNAs are removed with ______.
Without the __________ rnas cannot be _________

Answer 5

[is an improved method for] obtaining full-length cDNAs
[the] phosphate [groups from the] 5’ end [of] partial (uncapped) [mRNAs are removed with] alkaline phosphatase.
[Without the] free phosphate group [rnas cannot be] ligated to an adaptor

Question 6

3’ race
synthesize ______ from ______ by ________
Amplify using an ________ and a ________
To increase __________, you can perform __________ using a _______ and a _________

Answer 6

[synthesize] dDNAs [from] RNAs [by] reverse transcriptase
[Amplify using an] oligo-dT primer [and a] gene-specific primer
[To increase] specificity, [you can perform] another round of amplification [using] a nested gene specific primer [and a] primer for the oligo-dt primer