10.3 PCR Flashcards
If you heated the tube to separate dsDNA, what components of DNA replication would you no longer need?
- gyrase, helicase, DnaC, ori sequence, DnaA, single stranded binding proteins (nothing from initiation except template dsDNA)
If you added lab-produced DNA primers to the tube, what would you no longer need?
primase, RNA primer
If you only wanted short, linear pieces of DNA, what would you no longer need?
nothing from termination, as well as no sliding clamp/sliding clamp loader or ligase
What is needed to complete dna replication for PCR?
- template dsDNA/ssDNA
- DNA polymerase
- dATP,dGTP,dTTP,dCTP
- energy (ATP)
- and DNA primer
What was the problem with DNA replication in a test tube in 1975?
- 1 single stranded template creates 1 double stranded product
- DNA polymerase falls apart when the temperature iss raised, so new polymerase would need to be added to create a 2nd round of DNA synthesis
dNTP vs ddNTP
- dNTP= deoxynucleotide, contains -OH group
- ddNTP= dideoxynucleotide, -OH group is a H (molecule is reduced)
Sanger Sequencing (1977)
- a new method of sequencing DNA
- small quanitties of ddNTPs are added to a synthesis reaction. DNA synthesis is blocked whenever a ddNTP is incorporated into the new strand
- This results in synthesis mixtures containing different lengths of DNA, depending on where teh ddNTP was incorporated
- Only short sections of DNA can be sequenced at a time, cannot be multiplexed
the most accurate method of DNA sequencing
What can you do after sanger sequencing?
Run the dna pieces on a gel. Shorter fragments run faster than longer. Can tell where the fragments ended (which nucleotide). Then read the sequence to see the order of nucleotides.
What is used instead of sanger sequencing now?
- The same thing but with fluorescently labelled nucleotides
- Capillary tube used instead of a gel
What was so revolutionary about PCR?
you can make virtually infinite copies of dna
What did Kary Mullis discover in 1985?
- He used both a forward and reverse primer
- Realized if you use 2 primers, and a DNA polymerase that could withstand high temperatures, he could amplify the amount of dna synthesized exponentially
- Isolated DNA polymerases from bacteria that could withstand high temperatures
What are the 3 steps of a PCR reaction?
- Denaturation
- Annealing
- Extension
What happens during denaturation step?
Raise the temperature (~90C) until the dsDNA falls apart
What happens during annealing step?
- Lower the temperature just enought that the primers you’ve added will bind to the DNA sequence (~50C)
- But not so low that the whole double stranded DNA reforms
What happens during extension?
- Raise the temperature again to the temperature where that DNA polymerase that you’ve added is most optimal (usually ~72C)
- This is where DNA is actually synthesized
Why does PCR require a specific type of polymerase (for example, Taq polymerase)?
The polymerase must be able to withstand the range of temperatures in PCR without denaturing.
The function of the Polymerase Chain Reaction is to…
to create millions of copies of a specific region of DNA
What are restriction enzymes?
- there are hyndreds of these that have been purified from various bacteria
- they find a recognition sequence called the restriction site, which is where the enzyme binds and cuts the dsDNA
- some produce overhangs, others product blunt ends
What is the difference between endo and exonucleases?
- Endonucleases make internal (in the middle) cuts, exonucleases make terminal cuts
Dna pol 3 is ____. Restrictions enzymes are _____.
- Polymerase is when it adds nucleotides, nuclease is when it chews it up and destroys the nucleotide chain. When it goes back to fix mistakes, it is an exonuclease
- Restriction enzymes are endonucleases
Why would youadd a restriction site sequence to the 5’ end of the primers?
If its added to the 3’ end, you’ll interefere with its ability to actually prime a DNA reaction
What is 2nd gen sequencing (illumina)?
- allows you to sequence lots of DNA, all simultaneously in the same reaction
- localized PCR reactions allows for multiplexing
- Does this by chopping up a genome into little pieces (~150 bp per fragment), gluing those pieces in separate little parts on micro arrays in spots that hold different chunks of the genome, and use a localized PCR reaction to make more of those little chunks. Then youlet DNA polymerase add one nucleotide at a time w fluorescent probe and look to see what color got added
- high throughput
What is the result of illumina?
- Millions of little reads that needs to be put all together
- often use a reference genome to align sequences
Third generation sequencing: Nanopore
- long read sequencing (potentially up to a whole chromosome at once)
- DNA poly is embedded into a membrane. A strand of DNA is pulled through pol, and ionic current flow is measured to determine which nucleotide has been added
- Very sensitive and error prone
