Lecture 9 Flashcards
polymorphism
variation/ different versions of a trait/ allele in a population.
synthetic primer (oligo)
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
A
A or T
a
G or C
non synthetic primer
gives DNA a starting point
PCR
step1
-cool DNA or target so primer (oligo) can annel to target sequence
what do we not want in the primer?
repeated letters
what does genotyping let us do?
identify alleles (ex: sickle cell anemia comes from the presence of the GLU amino acid)
sanger method
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
dNTP vs ddNTP
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
sanger
-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)
modern technique
fluorescence with capillary electrophoresis (lets you test 800 to 1000 base pairs)
PCR
DNA replication in a test tube
-lets you amplify a sequence/ segment and replicate the same part on a massive scale
what does PCR use?
dNTP, DNA pol, primer (RNA if in vivo and DNA for in vitro) Mg 2+ for pol, and DNA template strand
what splits the DNA for PCR replication
heat NOT helicase
RNA pol in vivo?
lets DNA pol run/ unzip in the direction towards the fork/ bubble
PCR steps
heat to split DNA, cool to aneel the primer (one for the forward and one for reverse direction) and then heat for pol
what happens during first cycle?
You have two strands (lagging and template) that you cool so both primers can attach to their respective strands
what type of DNA pol so we use in pcr?
dna pol from an extremophile because the temperature would denature regular DNA pol
-most popular= Taq
where is the dna amplification from PCR visible?
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)
RFLP
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
sickle cell and RFLP
-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
old school RFLP
southern blot
how does southern blot work?
-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
RFLP vs Southern Blot
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)
what does electrophoresis in RFLP let us do?
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
whole genome sequencing
get all of the A, T, G and C of the chromosomes
-can be done by hierarchal approach or shotgun sequencing
