DB Lecture 28: RNA Analysis, PCR

Question 1

What is Nucleic acid hybridization?

Answer 1

the basis for detecting a particular mRNA being expressed in cell type

Question 2

Describe the process of Northern blotting.

Answer 2

1. remove of nuclei to prevent pre-mRNA contamination
2. using the poly(A) tail- hybridized to an oligo(dT) tract on a column
3. separate by gel electrophoresis on basis of size
4. transfer (blot) to a “membrane”
5. expose to labeled probe (sticks to target mRNA by virtue of sequence complementarity)
6. wash filter to remove excess probe
7. expose to X-ray film and record band pattern

Question 3

What information can be gained from a Northern blot?

Answer 3

tissue-specific gene expression
regulated expression
alternative splicing
deletion/insertion mutations
defective splicing
quantitative information about level of expression

Question 4

How do you do a PCR when your starting product is RNA rather than DNA?

Answer 4

convert the RNA to a DNA copy by reverse transcriptase then PCR reaction on the DNA

Question 5

What is a PCR reaction?

Answer 5

primer extension that copies template DNA starting from the free 3” hydroxyl end of a pair of primers
basis of almost all DNA diagnostics that will be an integral part of medicine

Question 6

What are the three temperature set points used during a PCR reaction?

Answer 6

denaturing (95 degrees C)
annealing (50-60 degrees C)
extension (72 degrees C)

Question 7

Describe what happens in the first cycle of PCR.

Answer 7

DNA is heated so that it splits into two single stranded pieces (denaturation) –> DNA is cooled and primers are added (annealing) –> thermoresistant DNA polymerase (Taq polymerase) is added and heated which extends each strand from the 3’ end of the primer (extension)
results in two long strands and two short strands

Question 8

Name the ways PCR can be used diagnostically.

Answer 8

detection of carrier for genetic diseases
detection of "foreign" genomes
single nucleotide polymorphisms (SNP)
PCR for RNA target (viral detection)
RNA Seq

Question 9

What factors are required for a PCR reaction to work?

Answer 9

template
primer
dNTPs
Mg2+

Question 10

Describe the subsequent steps of PCR after the first cycle

Answer 10

long and short strands are headed to remove from template DNA –> strands are cooled and primer and Taq polymerase are added –> elongation of primers on the new templates in the reverse direction, encompassing only the desired gene between the domains of the primers
leads to new templates that can be used in subsequent cycles to amplify the specific target sequence in a logarithmic function

Question 11

What is one example of how PCR can be used for carrier detection?

Answer 11

Tay Sachs
4bp insertion in Exon 11 OR 1 bp change (G to C) in Intron 12 (resulting in inclusion of intron 12 in the final sequence
If former: PCR with gel shows the insertion (two bands for carrier 4 kb apart)
If later: PCR (with endonuclease Ddel used prior) with gel shows one band at normal level and one band much lower

Question 12

How can PCR be used to detect a “foreign” genome?

Answer 12

targeting a known sequence on the organism (bacterial DNA or viral RNA) and using PCR for real-time detection of a PCR product (when coupled with cyber green fluorscence)

Question 13

How can PCR be used for SNP detection?

Answer 13

allows for characterization of a person’s genotype with respect to a particular polymorphism

Question 14

What is the pathway for using PCR for RNA targeted detection?

Answer 14

reverse transcriptase (RT) is used to convert mRNA into cDNA copy –> this can then be used in the standard PCR reaction

Question 15

What is RNA-Seq?

Answer 15

“NextGen sequencing”
able to convert mRNA isolated from a particular tissue into cNDA fragments (about 50 nucleotides from each fragment) allows for the creation of a cDNA “library” that maps the sequences in a transcriptome-wide fashion
also can be used to measure quantitatively the level of mRNA present for all expressed genes in that tissue