PCR Flashcards
PCR principle
selective amplification of a chosen region of a DNA molecule
Known sequences in PCR
those at the borders of the region
Short primers annealing to the template delimit
the region to be amplified
Amplification carried out by
DNA polymerase
PCR amplification is
exponential and non-stop
Nf
N0(1+Y)^n
n
of cycles
N0
initial copy # of target sequence
Nf
final copy # of amplified sequence
Y
efficiency of amplification per cycle
Steps in PCR cycle
- Denaturation of dsDNA template,
- annealing of primers
- Extention: synthesis of complimentary DNA by DNA polymerase
Sample PCR program
- denaturations 95˚, 30 sec
- denaturation 95˚ 30 sec
- annealing 55˚ 60 sec
- extension 72˚, 2.5 min
- Repeat steps 2-4 30 times
- Complete extension 72˚, 5 min
Problems in chaining
Solutions to the problems
Inactivation of DNA polymerase I during the denaturation step, Different temperatures required for three steps,
Thermostable DNA polymerases able to withstand multiple cycles of denaturation, Programmable thermocyclers that provide precise temperatures
Components of PCR
DNA template,
primer,
Taq polymerase and dNTPs
DNA template requirements
Contains at least one copy of target sequence
1 pg of plasmid DNA to 1 µg of mammalian DNA
ss- or ds- DNA
Linear or circular DNA
free of contaminants that may inhibit the reaction
Primer requirements
Quality (Automated DNA synthesizers, chromatographic or gel purification for more demanding applications)
Primer design is one of
the most important factors for successful pcr
Important factors in primer design:
length of the primer
having no significant homology with other sites on either strands of target DNA
not having self-complimentary sequences(within a primer or between primer pairs)
Primer Lengths Generally between
17 and 25
Shorter primers have increased likelyhood of
binding to non-targeted sites
Longer primers have higher ________ but lower ________
specificity, annealing rate
When annealing temperature is too high
primers and templates remain dissociated
When annealing temperature is too low
There are mismatched hybrids and not all of the correct bp form
With correct annealing temperatures
Priming occurs only at the desired target sites
Tm
4 * (G+C) + 2*(A+T)
Tm’s for two primers need to be similar in order to
minimize the potential for mispriming
optimal base compositions
~40-60% [G+C] content
Annealing temp typically
~2-5 ˚C below Tm
Annealing time depends on __________ but a ____________
length and complexity of the primers, fixed time is usually used
Thermostable DNA polymerases are
isolated from thermophilic and hyperthermophilic archaebacteria, stable at high temperature, have a tradeoff between extension speed and proof-reading function
Taq DNA polymerases
have faster extension
are more tolerant of inhibitory substances
tend to add a overhanging adenosine
Pfu polymerases
lower error rate due to 3’-5’ exonuclease activity
can amplify longer fragments
stronger thermal stability
Ways to visualize and Identify PCR products
by size restriction analysis (Sites present in amplified DNA, Mutations) by sequencing (purification, template for sequencing)
In T-A cloning, taq polymerase tends to
add a non-templated A to the 3’ end
In T-A cloning, cloning is into a special vector with
“T” overhangs(TA vector) for base-pairing with the ends of the pcr product
polynucleotides synthesized from taq polymerase often have an
extra adenosine at their 3’ end
In preparing a TA vector
Treat blunt cut vector with ______
Engineer a vector containing _____
Taq in the presence of dTTP
an Xcml site
In cloning by restriction sites
use _____________ in the DNA target
introduce __________ at the _______ of the PCR primers
Newly created sites must be _______________
naturally occurring restriction sites
new restriction sites at the 5’ end of the PCR primers
absent from the amplified DNA fragment
Allelle specific pcr There is at least \_\_\_\_\_\_\_\_ Design a primer having \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ the mismatch is at \_\_\_\_\_\_\_\_\_\_\_\_ Use a polymerase w/out \_\_\_\_\_\_\_\_\_
one nucleotide difference between the two templates
a perfect match with one allele, but a mismatch with the other allele
the last nucleotide
3’ -> 5’ exonuclease activity
Taq can only amplify fragments up to
3-5 kb
Long PCR
Combining two DNA polymerases allows
Has an _____ but _______ “workhorse to _________
has small amounts of ________ with ___________ which removes ________
amplifation of DNA fragments up to 20-30kb
efficient, error-prone, carry out most of the DNA synthesis
proof-reading polymerase, 3’-5’ exonuclease activity
, mismatched bp
Assembly PCR
synthesize longer DNA from two paritally overlapping primers or DNA sequences
Gibson assembly
synthesize longer DNA from two or more overlapping primers or DNA fragments, cloning without restriction sites
RT-PCR
determine the amount of RNA transcripts using cDNA as the template
multiplex PCR
Apply two or more targets in the same reaction
BRIDGE PCR
DNA fragments are ____________
Has a solid surface coated with __________
First round of synthesis results in ________
With nucleotides and polymerase added ____
The end result is
- ligated w/ 2 adaptors
- 2 types of primers corresponding to the adaptors
- newly synthesized DNA having the 5’ end bound to the solid surface
- further amplification is realized only when anchored DNA forms a ‘bridge’ with a primer on the surface
- localized amplification of a single molecule of DNA template
Emulsion PCR, prepare a ________ by preparing an ________ containing _________ and adding the _______ to ________ while _________
water/oil emulsion
- preparing an oil phase (w/ surfactant)
- preparing an aqueous phase containing pcr products
- aqueous solution to oil phase while stirring is taking place
