Unit 9: PCR strategies
What is the starting material for PCR?
1.) Template DNA ; contains regions that needs to be amplified. 2.) Oligonucleotide primers ; complementary to the ends of the DNA fragment to be amplified. These are synthetic and about 15-20 nucleotides long. chosen by the researcher. 3.)Deoxynucleoside triphosphates (dNTPs) ; provide the precursors for DNA synthesis 4.) Taq polymerase - KEY "ingredient" ; DNA polymerase isolated from the bacterium Thermus aquaticus. This thermostable enzyme is necessary because PCR involves heating steps that inactivate most other DNA polymerases. Buffer with magnesium.
What are some features of optimal primers for PCR?
18 - 24 nt 50-60 % GC avoid palindromes, complementation of primers (no primer dimer), repeat sequences (blast to make sure it is unique)
A typical PCR is likely to involve how many cycles of replication? How long does this take?
20 to 30, this takes a few hours to complete
Assuming 100% efficiency, how much does the target DNA increase after 20 cycles of PCR? What about 30 cycles?
After 20 cycles, a target DNA sequene will increase 2 ^20 fold (~1 million fold) After 30 cycles, a target DNA sequence will increase 2^30 fold (~1 billion fold)
Which of the following is not needed for PCR? DNA polymerase all four nucleotides target DNA DNA ligase Primers
DNA ligase
What are the steps in PCR?
Denaturing: 94-95 degrees Celsius Annealing: (of primers) Temp ranges based on the primer set that you use. 45-60 degrees Celsius Synthesis (extension): 72 degrees Celsius, the tempt hat the bacteria (thermus aquatus) lives at. This is repeated many times
What are the three phases in real time PCR?
Exponential phase: proportional to the amount of starting material Linear phase: reaction efficiency falls, reagents become limiting Plateau: one or more reagents used up (note: number cycles (exponential phase) to reach cycle threshold (use standards).
Who developed PCR? I M P O R T A N T
Kary mullis `
Inverse PCR
Method for using PCR to amplify unknown sequences by circularizing the template molecule
What is a limitation of PCR?
Part of the target DNA sequence needs to be known.
In PCR, how can you avoid contamination?
Primary: aerosol barrier tips (or filter tips) Have separate pipettors for the DNA vs. the PCR reagents. You can put the pipettors under UV light because that causes thymine dimers and then the DNA polymerase cannot get through the thymine dimers. So, even though there may be DNA there, it will not amplify it. Set up PCR in one room and the DNA in a different room.
Lets say, you want to know more than just the internal piece of DNA and you want to know what the 3' ends look like, or maybe both ends. What do you use?
R A C E Rapid amplification of cDNA ends
How is RT-PCT carried out? (reverse transcriptase PCR)
RNA is isolated from a sample It is mixed with reverse transcriptase and a primer that will anneal to the 3' end of the RNA of interest. This generates a single-stranded cDNA which can be used as template DNA in conventional PCR.
What is an alternative to real time PCR (qPCR)?
SYBR green. This binds to the minor grooves of dsDNA. So, the more copies of dsDNA you get the more SYBR green that will be bound to DNA. When it binds to DNA it fluoresces more. Pro: the cost Con: The resolution is not the same as Taqman
What is the key component of PCR?
Taq polymerase
Reverse transcriptase PCR is used to detect and quantitate what?
The amount of RNA in living cells.
Explain the cycle threshold method in real time PCR (qPCR).
The cycle threshold reached when accumulation of florescence is significantly greater than the background level. Early cycles background levels are greater than that dervied via PCR. Once the cycle threshold value (ct) is exceeded, the exponential accumulation of product can be measured (compared to a standard)
The ___________ molecule blocks the fluorescence of the _______ molecule on the oligonucleotide.
The quencher molecule blocks the fluorescence of the reporter molecule on the oligonucleotide.
PCR is carried out in a ______, which automates the timing of each cycle
Thermocycler
What is a primer dimer in PCR?
This is where the primer is binding on itself and amplifying.
In PCR, what will you see if you have too much or too little MgCl2 (magnesium chloride)?
Too much: Impacts primer annealing, if the primer does not anneal right then you will get more primer dimer artifacts. Too little: Your enzyme activity will decrease and you will get low yield.
In PCR, what will you see if you have too much or too little Dntps?
Too much: Misincorporation (might get a A slip in where there should be a T) get incorrect sequence (lower fidelity) Too little: Low yield, you do not have enough precursors so not good yield.
In PCR, what will you see if you have too much or too little Taq?
Too much: More background product / artifacts. Too little: low yield
In PCR, what will you see if you have too much or too little template?
Too much: You will get miss priming, there is so much template that your primers might bind to the wrong places. Too little: Your yield will be low
What is the outcome of PCR?
You generate millions of billions of copies based on the known sequence.
Rapid Amplification of cDNA Ends (RACE)
You need the 5' end You take a primer based on your unique sequence and then a primer based on a sequence going the other way. Then you would amplify and have the 3' end. You are essentially a primer beyond the alago t. RACE shows you that you can use RNAse H to degrade the RNA.
In real time PCR, what can be used as a control?
beta actin. Could even be a plamsid that has a certain gene it it. You want something that will be constant among different tissue types.
PCR (polymerase chain reaction)
in vitro amplification of DNA- a way to copy a specific sequence of DNA without vectors and host cells (alternative to cloning)
Real-time PCR (qPCR)
is carried out in a thermocycler that can measure changes in fluorescence emitted by detector molecules in the PCR reaction mix. It can be used to quantify the amount of DNA or RNA present in a specific gene or mRNA. The TaqMan system uses a detector oligonucleotide that has fluorescent reporter molecule at one end and a quencher molecule at the other end. The quencher molecule blocks the fluorescence of the reporter molecule on the oligonucleotide. Taq polymerase 5' - 3' exonuclease activity digests the probe separating reporter and quencher. -fluorescence detected by thermocycler will increase in proportion to the amount of PCR product produced.
Is PCR a replacement to cloning?
no, this is because you have to know enough about the gene. You have to know the sequence already in order to amplify it.
in order to amplify DNA, you need _______?
primers
Why is Taq polymerase used in PCR?
stable at high temperatures Taq (thermus aquatus) live in hot springs. In order to amplify DNA, you have to separate the strands by denaturing at a high temperature, however, you do not want to denature the polymerase. Since taq polymerase live in hot springs, it impacts them less.
In PCR, why do you run a negative control?
this is because you want to ensure that there is no contamination. If you are amplifying something millions of billions of times, the slightest bit of contamination will be seen. (the pipettors themselves is where most of the contamination comes from) The negative control is all of your reagents except the DNA. You will ge3t primer dimers but should get no product.
In PCR, what will you see if you have too high or too low annealing temp?
this is the easiest to change! too high: you will not get product because primers won't anneal. too low: might get mispriming. This would yield artifact bands.
How is real-time (qPCR) quantitated?
via cycle threshold method.
