exam 3: DNA at High Resolution

Question 1

Once you have a library, how can you find a sequence?

Answer 1

screen library via hybridization

Question 2

Library screening: Hybridization

Answer 2

-transform host cells with ur lib and plate on an antibiotic to screen for host cells that have your vector

-place nylon/ nitrocellulose on membrane (+) on colonies and “lift” them from plate=colony lift
—> Some cells will stay on a plate (& stay alive) why? To reproduce
—-> some stick to membrane
*why? work w membranes

-lyse open cells attached to membrane; DNA (-) will bind to filter (by changing pH)

-chemically treat membranes to make bound DNA Single Stranded

-making a probe: synthesize the known sequence related to the sequence of interest + label it with a visible marker (radioactive/ fluorescent tag)

-incubate membranes with probe to allow complementary sequence to hybridize (bind)

-wash away the unbound probe & look for visible signs of the bound probe

-go back to plate that had colony that bound to probe and grow more cells from this colony
—>what can you deduce abt fragment cloned in vector in these host cells? has the gene ur looking for (gene is present in organism)

Question 3

what is a probe?

Answer 3

a short, single-stranded piece of DNA that is used to hybridized (bind to complementary sequence

Question 4

other uses of hybridization (Southern plot)

Answer 4

-can be used to detect similar sequences in any collection of nucleic acid fragments (detect the presence/absence of a sequence)

example:
Southern plot
-can cut genomic DNa w REs
-run through agarose gel
-transfer cut DNA fragments to membrane
-hybridize w labeled probe (complement to what your looking for)

+procedure is exactly the same as library sceen, but no molecular cloning
-why not just run southern blot? why build lib?
=>benefit of molecular cloning
1) have it isolate: able to run experiments
2) in living cells: reproduce + make copies of dna-> have fragments in living host cell

**Northern Blot: RNA
**Western Blot: Protein
**Southwestern Blot: DNA binds to Protein

Question 5

Polymerase Chain Reaction (PCR)

Answer 5

-amplification of specific region of DNA
-must know sequence of DNA flanking target region to be amplified

3 steps:
denaturation
annealing
extension

*repeat 3 steps 20-30 times

+every round of PCR double # of copies of target DNA
(2 template strands for DNA double strands)
(4 template strands for 2 DNA double strands)

Question 6

PCR: denaturation

Answer 6

(high heat)
start double strands, denatured by high heat to separate into 2 separate single strands (95C)

Question 7

PCR: annealing

Answer 7

(low heat)
lower the temp to allow binding of primers (short single-stranded pieces of DNA complementary to region flanking target DNA) thru hydrogen bonds

Question 8

PCR: extension

Answer 8

(68-72)

raise temp to allow DNA polymerase to copy template/target DNA

*elongate, DNA polymerase reads template + put in complementary bases (Taq)

Question 9

PCR: what do you do with PCR fragments?

Answer 9

+clone into vectors using molecular cloning
+sequence
+use in electrophoresis to look for size differences

Question 10

*advantages of PCR (be able to tell 2)

Answer 10

1) requires very little DNA; only need 1 copy of template DNA (to double amt of DNA)

2)fast, only takes a few hours

3)works on degraded DNA; as long as primer sequence is not degraded

Question 11

*disadvantages of PCR (know 2)

Answer 11

1) requires knowledge of sequence flanking target DNA (primers)
-have to know sequence that flanks it (pieces around it)

2)can only amplify small fragments (<25 kb)
-cannot look at multiple+ chunky sequences

3) does not include the entire genome, very specific
(can be adv if only want to look @1 specific sequence

Question 12

what is sequencing

Answer 12

determining the order of bases of DNA in the 5’ to 3’ directiom

how is this useful?
-can draw RE maps (estimate the distance where RE can cut)

-can predict amino acid sequence of protein-coding genes

-can screen for mutations ( screen ppl dont hsve disease/disorder and those dont)

-what changed overtime?
-compare w/ known sequence

Question 13

*know the difference
in what ways are sequencing different from PCR?

Answer 13

sequence:
-only 1 primer is used (read off only 1 strand @ a time)
-dNTPs & ddNTPs (PCR: DNTPS)
->1 OH on 3’

+ddNTPs(dideoxy): no 2’ or 3’ OH group
->what happens to a growing DNA polymer if a ddNTP is incorporated?
=stop the rxn: length of fragment will be whatever length when ddNTPs add (no base can no longer be added)

Question 14

Process of setting up sequencing

Answer 14

1) set up a4 separate rxn : each rxn has
-all dNTP’s (including one that is labeled w a visual marker)
—-+ radioative isotope & 3’ OH group
-1 primer complementary to the region flanking the target DNA (just sitting outside what u want to sequence)
-DNA polymerase
-1 of the four ddNTP’s (G A T C:only 1 used)

Interpretation:
-create a series of fragments of variable length that end in ddNTP
-length determined by where ddNTP was incorporated
-run 4 rnx in separate lanes on polyacrylamide gel (why? size dif<30 bp)
-read order of bases based on length
-able to visuallze bc 1 of the dNTPs has visible label (once have all fragments in size, use electrophoresis to separate)

**look at ppt to review

Question 15

automated sequencing

Answer 15

-works the same way as traditional sequencing, but all 4 ddntps used in same rxn

-label 4 ddNTP’s w unique nonradioactive flourescent markers to distinguish between them (4 diff colors)

-run on automated sequencer (laser scans fragments to determine flourescent color)

-specific color band coresspond to specific ddNTP associated w that color