RNA Analysis, PCR- Lecture 28 Flashcards
What is an oligo?
a DNA oligonucleotide that is normally a sting of about 18-25 nucleotides that is complementary to a sequence on mRNA with a 5’ radioactive phosphate group for labeling (probe)
Describe the Northern blotting procedure.
isolation of mRNA is added to a gel and electrophoresis is used to separate on the basis of size –> mRNA can be transferred (blotted) to a “membrane” –> membrane exposed to labeled probe (sticks to target mRNA b/c complementarity) –> wash filter (removes excess probe) –> expose to X-ray film and record banding pattern
Describe the process of isolating mRNA.
remove nuclei –> pass cytoplasmic mix of RNA through oligo-dT column (mRNA polyA tail sticks while everything else washes through) –> wash –> elute column in low salt buffer to gain solution of purified mRNA
What information can Northern blotting provide you?
information about tissue-specific gene expression, regulated expression, alternative splicing, deletion/insertion mutations, and defective splicing
quantitative information about the level of expression
Describe how you would need to prep PCR for RNA PCR replication rather than DNA.
RNA is converted to cDNA by reverse transcriptase –> PCR reaction is used on DNA copy
What is PCR?
a primer extension that copies template DNA starting from teh free 3’ hydroxyl end of a pair of primers (short DNA oligos complementary to the DNA sequences some distance apart)
What is the usefulness of PCR?
it amplifies the target sequence billions-fold
What are the three temperature control set points employed in the PCR reaction?
Denaturation- 95 degrees C
Annealing- 50-60 degrees C
Extension- 72 degrees C
Describe what happens in the first cycle of PCR.
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)
this results in two long strands and two short strands
Name the ways PCR can be used diagnostically.
detection of carrier for genetic diseases, detection of “foreign”genomes, single nucleotide polymorphisms (SNP), PCR for RNA target (viral detection), and RNA-Seq
What factors are needed for a PCR reaction to work?
template, primer, dNTPs, and Mg2+
Describe the subsequent steps of PCR after the first cycle.
long and short strands are heated to remove from template DNA –> strands are cooled and primers 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
this leads to new templates that can be used in subsequent cycles to amplify the specific target sequence in a logarithmic function
How is PCR used for carrier detection?
PCR amplifies particular sections of chromosomes where point mutations or small “indels” are known to be located
What is one example for the use of carrier detection with PCR?
Tay Sachs is know to be caused by either (1) 4bp insertion in Exon 11 or (2) 1 bp change (G to C) in Intron 12 (resulting in inclusion of intron 12 in the final sequence)
(1) PCR with gel shows the insertion (two bands for carriers 4 kb apart)
(2) PCR (with endonuclease DdeI used prior) with gel shows one band at the normal level and one band much lower
How can PCR be used to detect a “foreign” genome?
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 florescence)
