PCR
Requirements for DNA Replication (5 things)
-DNA template; -Primer (starting point for new strand of DNA) -Nucleotides (A, G, C, T) - "dNTPs" -Mg++ and other ions/ buffer (salt + tris) -A thermal stable DNA Polymerase (to catalyse)
First step in PCR Process
1. Denature DNA to ssDNA by heating to 95 deg F
Benefits for PCR-Based Methods
1. fast (1-2 days) from extraction to typing results (fewer personnel and simple interlab comparison); 2. less DNA required (0.1-1ng); 3. degraded DNA can be analyzed (smaller target regions); 4. discrete allele values obtained; 5. Many loci analyzed simultaneously (multiplexes and megaplexes) 6. automatable
Primers
18-30 nucleotides long stretch of DNA; synthetically created; flanks the region of interest: must be unique ("assigned") to region, used in pairs; platform for DNA polymerase to make new DNA
G/C content in primers
50% GC
G/C Clamp (primer selection)
A G/C at the 3' end can strengthen bonding
TaqGold and other hot-start polymerases are inactive until activated by heat (part 2):
Add all reagents including Taq into amplification reaction; 95 deg C for 10' changes structure to active enzyme - prevents nonspecific amplification
Second step in PCR Process
Annealing; Temperature is raised to 55-70 deg F; DNA primers that match sequences flanking the locus of interest anneal
Polyadenylation with Taq
Certain polymerases add 1-2 A's to the end of the amp product during extension step
PCR Cycles (3)
Denaturation, Annealing, Extension; Each cycle results in a doubling in the number of copies of the target region
Third step in PCR Process
Elongation; Temp raised to 70-72 deg F; DNA Polymerase extends the primers- adds new nucleotide to produce a copy of each DNA template
How do you denature DNA without killing (denaturing) the enzyme?
Find an enzyme that can take the heat
Polymerase makes new strand with a primer
In vivo: RNA, primase primer will be complementary; need a pair of primers if you want defined size or logarithmic amplification;
PCR Prep- placing samples in order
Negative amplification control (no DNA template added; just amp reaction chemicals) Last sample to be prepared; Tubes open during PCR step process
Fourth step in PCR Process
New copies of DNA strand are made
7 Common PCR Inhibitors
Phenol, heme, competing DNA's, dyes, leather, beech wood, EDTA`
Thermal Cycler
Programmable heating and cooling unit
Fifth step in PCR Process
Repeat the process 25-30 times; exponential increase in copy #; after 30 cycles, approximately 1 billion copies
DNA Template
Sample DNA extract contains entire genome in multiple copies for the individual or organism being studied; need 0.5-2ng of template DNA in reaction for forensics
DNA Polymerase- Taq
Thermus aquaticus; Lives at 50-80 degrees Celsius
Working formula for Primers
Tm= 4 deg C x (number of G's and C's in the primer) + 2 deg C x (number of A's and T's in the primer)
Selection of primers made by using...
a known sequence of target region and primer selection software
STRs are sensitive to single bp changes- solution:
add 30' extension step at 72 deg C at end of PCR reaction (ensures that A's are added consistently to each PCR product)
PCR Prep- Creating master mix
all reaction chemicals (minus DNA template) in sufficient quantities for the number of samples; pipette master mix in each tube and change tips in between; Add DNA template to reaction tubes LAST
Non-specific Amplification
amplification of DNA sequences other than target sequence; results in multiple amplification products
Potential causes of non-specific amplification
annealing temperatures too low; poor primer selection; Taq polymerase activity outside PCR cycles
Nucleotides (role in PCR)
at least 200 uM of dATP, dCTP, dTTP, dGTP; equal concentrations of each nucleotide; building blocks for new DNA strands
Each locus has power of discrimination; however, multiple loci...
can be analyzed simultaneously, making the process faster; independent, unlinked loci; with 8-13 loci tests, same P
QC for PCR: Pre-PCR prep
clean area with 10% bleach followed by isopropanol or EtOH; PCR reactions set up in a laminar-flow hood or other separate work area; Dedicated pipettors and lab coat, change gloves often
Mg++ ions
cofactor for Taq Polymerase ; optimum concentration varies for each primer set (0.5-2.5mM); added separately; essential co-factor at active site for catalyzing reaction
Reverse primer
complementary to forward strand; reverse complement of end of sequence
Forward primer
complementary to reverse strand
Primer sequences are ____ to target strand; become part of the ____.
complementary; PCR fragment
PCR History
described by Kary Mullis in 1985 1993- got nobel prize in Chemistry for PCR
PCR Specificity
determined by the primers; optimally specific for only one target
PCR replication is a ____ process, thus ____ .
doubling; minute/tiny amounts of DNA can be made into virtually unlimited quantities
PCR type of rxn
enzymatic reaction to amplify specific regions of DNA (target seq.)
Buffer
maintains optimal salt concentration for enzyme activity; maintains optimal pH; salt + tris + water = ammonium sulfate -> phosphate backbone (-)
PCR
method for using the cell's own machinery for in-vitro DNA replication; can make millions to billions of copies of target DNA in hours
Selection of Primers
must be a unique sequence; prevent complimentary primer-pair sequences no inter-primer sequences (primer-dimer); No intra-primer sequences (hairpin loops)
Normal Taq Polymerase is always active (part 1):
pipette all reagents EXCEPT for Taq into the amp reaction; perform Hot-Start (95 deg C for 5'); Add Taq; Start PCR reaction
Avoid ____ stretches in primer selection
poly dNTP stretches
Melting temperature of primers (Tm)
primer pair designed w/ similar Tm; best b/w 55-72 deg C;
Standardized results of PCR
same loci used across the world; simple interlab comparison
QC for PCR: Post-PCR work area
separate area (preferably different room); PCR reaction product NEVER goes into evidence handling/ extraction/ quantitation/ pre-PCR areas
Length of amplification product
usually 100-600 base pairs
