11.2 PCR (did someone say?) Flashcards
why is qPCR the gold standard for any disease?
extremely accurate but require special equipment
What are the requirements for PCR?
- DNA template (single or double, doesn’t need to be pure)
- thermostable DNA polymerase (taq)
- DNA primer (usually 2)
- dNTP: dATP, dCTP, dGTP, dTTP
- Mg2+ : essential for enzyme, affects annealing
others: salt, stabilizers, pH control
describe the polymerase chain reaction? Who discovered it?
-mullis, 1983
- discovered that if two primers were used that aligned with the opposite strand, that could lead to exponential DNA increase
- “the most important technique in molecular biology”
how many cycles are in PCR? What does this mean for the number of DNA copies?
- 30-35 cycles before materials run out
- theoretically (assuming 100% efficiency) over a billion copies
what are the steps and temperatures for PCR
- denaturation: 94-96 degrees, double stranded DNA split into single strand
- annealing: 50-65 degrees, primers bind, Tm dependant on the length and base composition of primers
- elongation : 72 degrees
- DNA polymerase binds to annealed orients and extends DNA In 5’ to 3’ direction
how is temperature cycling performed ?
- using computer controlled heating/cooling devices
- thermal cycler / PCR machine
describe the primers in more detail?
- primers are short molecules of single stranded DNA (oligo nucleotides) often 18-25 bp long (may be shorter/longer)
- size of PCR depends on how far annealing ends of the primer are
-PCR products of up to 40 kb have been produces but more common is <2 kb
What is the relationship between length of primers and yield of PCR?
- yield drops with increasing primer length
why are primers generally 18-25 bp long?
- specificity of primer binding is related to primer length
- short primers may not be specific enough, bind to too many places
- longer primers are slightly more accurate but more costly
- primers 18-25 bp long are USUALLY long enough to match only to their intended sequence
what are some application of PCR?
- amplification of target sequences for further study : amplifying target from within a complex mixture is equivalent to purification - could amplify gene for further study
- detection of rare DNA sequences : can detect as little as a single copy of DNA
- bacterial food contaminants, bacteria etc. (not ideal for determining the amount though)
What is PCR not useful for?
determining the abundance of these sequences (only their presence)
- so we can say that YES, this bacteria is present but we have no idea how much we started with
what are the phases of a PCR graph?
- exponential (doubles every time)
- when below the threshold this does NOT mean no duplication is occurring, just that it is not yet detectable
slower growth - linear phase
eventually dNTP and polymerase becomes exhausted.= plateau
what provides the best estimate to the starting amount of DNA (or RNA) in a template?
log linear phase
How is DNA quantified in each cycle most commonly?
- growth in amount of PCR product is monitored using reported dye and a PCR
machine - SYBR gene is simplest/cheapest reporter dye (intercalating) : can be re-used
- SYBR fluoresces greatly when bound to double stranded DNA, binds primarily to minor groove in double strand
what is another method of quantifying PCR?
- fluorescent probes that come in different colours: can track multiple reactions using different colour for each target
What are some applications of qPCR?
- quantify the starting amount of a particular sequence
- measuring the rate at which a particular gene is transcribed
how can qPCR be used to measure gene expression?
the amount of PCR product (exponential) is proportional to the amount of starting DNA
