Ch. 7 - Nucleic Acid Amplification

Question 1

RT-PCR

Answer 1

Reverse transcription polymerase chain reaction
Used to qualitatively measure mRNA
Very sensitive

Question 2

What are the 5 steps in RT-PCR?

Answer 2

1. Isolate mRNA
2. Use a primer to make cDNA to the mRNA
3. Add RNase to get rid of the RNA strand (only want cDNA)
4. Add primers + DNA polymerase to convert ssDNA to dsDNA
5. dsDNA replication via PCR

Question 3

cDNA

Answer 3

Complementary DNA

Question 4

Total RNA –?-> mRNA –?-> mRNA/cDNA –?-> ss cDNA –?-> ds cDNA –?-> many ds cDNA

Answer 4

isolate via polyT or polyU 
primer, reverse transcriptase 
RNase
primers, DNA polymerase 
PCR

Question 5

Northern blot

Answer 5

Detects RNA using a radioactive (32P) or chemical probe

Question 6

What are the 6 steps in a N. blot?

Answer 6

1. mRNA extraction (polyT or polyU)
2. Electrophoresis
3. Transfer to membrane
4. Fix mRNA via heat or UV
5. Incubate membrane with labeled probes
6. Visualize with x-ray film to see bands

Question 7

Which is more sensitive, N. blot or RT-PCR and why? Which is more commonly used?

Answer 7

RT-PCR is more sensitive because is can detect very low levels of mRNA; it is more commonly used.

N. blot is less sensitive as it cannot detect very low levels of mRNA, and is therefore less commonly used than RT-PCR.