DNA sequencing methods

Question 1

what is southern blotting

Answer 1

this is taking DNA and cutting it up into pieces using RESTRICTION ENZYMES. and then running it on an agarose gel. going to have to run with nuclei acid probe so that you can find fragment of choice.

Question 2

what is PCR

Answer 2

PCR is a way to amplify DNA
so you melt the strands and use a primer that is specific for a certain region
Add TAQ POLYMERASE enzyme which will copy DNA btwn the 2 pieces.
melt and repeat to amplify DNA

Question 3

what is dna sequencing?

Answer 3

you take the dna, and melt it.
then run it with taq polymerase,but use dideoxynucleotides so that the 2’ OH doesn’t have OH for next one to add. this makes each one longer than the previous fragment.

Question 4

what is a northern blot

Answer 4

begin with cell with different types of RNA( t, r,m) and then denature
run on gel with formaldehyde (these suckers irk to anneal/fold) and add complementary probe.

can (unlike souther) quantify amount: by intensity of signal
-can also analyze splicing

Question 5

what is RT-PCR?

Answer 5

reverse transcriptase PCR: mRNA with poly a tail and 5’ cap.
PRIMER on poly a tial

PCR needs to be double stranded so add reverse transcriptase which will copy from mRNA. but then it degrades the original mRNA and then recopies the original copy to get dourble stranded.

this db stranded is now cDNA

Question 6

what is microarray?

Answer 6

where you take lots of PROBES, like thousands. and put them on a slide. then take mRNA and let it hybridize. you will see intensity to tell you how many specific matches are there

Question 7

what is the difference btwn souther, northern, and western blot?

Answer 7

western: uses protein, use denaturing gel but its called ACRYLAMIDE, and then use antibodies to mark proteins
northern: use RNA, and use denaturing formaldehyde gel and use complementary probe marker of nucleic acid

Southern blot: do DNA, use gel and then use complementary probe marker of Nucleic acid

Question 8

where do you get dna for Souther blot.

what are the steps?

Answer 8

1) DNA from cheek swab to get peripheral blood leukocytes,
2) use detergent
3) restriction enzymes cut (polymorphisms alter enzyme recognition)
4) separate by gel electorphressis where DNA runs to opposite end
5) capillary transfert to nitrocellulose
6) hybridization with label that bind complementary sequence
7) can see alleles by cuts of RFLP