Lecture 9

Question 1

polymorphism

Answer 1

variation/ different versions of a trait/ allele in a population.

Question 2

synthetic primer (oligo)

Answer 2

DNA sequence that complements the target sequence/ DNA you are mapping
-denatures the target DNA
-50% is GC (if it were less it would aneel wrong to the target DNA

Question 3

A

Answer 3

A or T

Question 4

a

Answer 4

G or C

Question 5

non synthetic primer

Answer 5

gives DNA a starting point

Question 6

PCR
step1

Answer 6

-cool DNA or target so primer (oligo) can annel to target sequence

Question 7

what do we not want in the primer?

Answer 7

repeated letters

Question 8

what does genotyping let us do?

Answer 8

identify alleles (ex: sickle cell anemia comes from the presence of the GLU amino acid)

Question 9

sanger method

Answer 9

sequencing with dideoxy chain termination method
-tells us the letters in order in DNA sequence which lets us determine amino acids
-ddNTP is modified to attach to synthetic DNA of different lengths and has 4 flourescent tags

Question 10

dNTP vs ddNTP

Answer 10

dNTP has a 3 prime hydroxyl while ddNTP has a 3 prime hydrogen
-ddNTP has no phosphorous therefore it can stope the chain and keep it short
-dNTP has phosphorous so it attaches to corresponding base and keeps going

Question 11

sanger

Answer 11

-added ddNTP to stop strands at specific points
-EX: ddA stops the strand at the A codon, ddC at the C codon and so on
-this is then put into gel electrophoresis and the longer they run on the gel the shorter the fragments of DNA are
-at the end you know the size of the DNA (from the gel) and the sequence (from the ddNTP)

Question 12

modern technique

Answer 12

fluorescence with capillary electrophoresis (lets you test 800 to 1000 base pairs)

Question 13

PCR

Answer 13

DNA replication in a test tube
-lets you amplify a sequence/ segment and replicate the same part on a massive scale

Question 14

what does PCR use?

Answer 14

dNTP, DNA pol, primer (RNA if in vivo and DNA for in vitro) Mg 2+ for pol, and DNA template strand

Question 15

what splits the DNA for PCR replication

Answer 15

heat NOT helicase

Question 16

RNA pol in vivo?

Answer 16

lets DNA pol run/ unzip in the direction towards the fork/ bubble

Question 17

PCR steps

Answer 17

heat to split DNA, cool to aneel the primer (one for the forward and one for reverse direction) and then heat for pol

Question 18

what happens during first cycle?

Answer 18

You have two strands (lagging and template) that you cool so both primers can attach to their respective strands

Question 19

what type of DNA pol so we use in pcr?

Answer 19

dna pol from an extremophile because the temperature would denature regular DNA pol
-most popular= Taq

Question 20

where is the dna amplification from PCR visible?

Answer 20

Gel electrophoresis
-shows the true alleles because an allele w an indel/ mutaiton will be a different size than we expect
(ex: sickle cell shows the indel which causes missence in the gel)

Question 21

RFLP

Answer 21

region to amplify/ sequence for mutation
-restriction fragment cut long complex DNA at sugar phosphate bonds
-the pieces vary in length bc everyones DNA is different
-comparing the different lengths can show different sequences/ mutations
-this is easier if the mutation creates a restriction site

Question 22

sickle cell and RFLP

Answer 22

-sickle cell does not have the Dde1 restriction site like wild type therefore it does not let the restriction enzyme cut where wildtype would
-this happens because the mutation makes a A turn into a T in the code sequence
-therefore the sickle cell DNA will be a different length than wild type after it is cut

Question 23

old school RFLP

Answer 23

southern blot

Question 24

how does southern blot work?

Answer 24

-put the DNA and the restriction enzyme in tubes so the whole sequence is digested then run it on gel
-use filter paper on the gel and the DNA will stick to it because of the nitrocellulose in the filter paper
-use a radioactive probe of the gene we want to unwind the DNA on the paper and base pair it with the probe
-because the mutation base pairs with the probe only you can look under x ray to see where the mutaiton is

Question 25

RFLP vs Southern Blot

Answer 25

RFPL tells us the exact order of the letters int he DNA (like order of letters in a word) while southern blot finds a specific piece of DNA in a sample (detects a mutation does not read the code letter by letter)

Question 26

what does electrophoresis in RFLP let us do?

Answer 26

Put the genetic code in order. Once the restriction enzymes cut the DNA into tiny pieces we need to put the letters together to get the letter by letter code. Based on how fast they run on the gel we can esitmate their position in the sequence

Question 27

whole genome sequencing

Answer 27

get all of the A, T, G and C of the chromosomes
-can be done by hierarchal approach or shotgun sequencing