exam 2 | part 2 - nucleic acid amplification

who developed PCR?

chemist Kary Mullis

synthetic oligonucleotides

1993 Nobel Prize in chemistry

purpose of PCR

to amplify original DNA sample

amplicons

each copy made from original PCR sequence (products)

how does PCR work?

continuous amplification of template by repeating cycles of replication

total product (amplicon)

2^N

N = # of cycles

basic PCR procedure

DNA replication in vitro

template

master mix

what is contained in a master mix?

dNTPs

DNA polymerase

primers

buffer

dNTPs (deoxyribonucleotide bases)

monomers, the building blocks of the growing strands

DNA polymerase

catalyze rxn by adding the dNTPs

primers

set of oligonucleotides

indicate DNA pol starting point

buffer

Tris, pH 8.4

MgCl2

KCl

PCR steps

denaturation

annealing

extension

temp for denaturation

90-96 C for 20-60 sec

temp for annealing

50-70C for 20-90 sec

temp for extension

68-75C for 10-60 sec

denaturation

GC content is important

need warmer temps + longer time in this step because there’s more H bonds to break

annealing

temp and time critical for PCR specificity

extension

DNA synthesis takes place through the pol synthesizing copy of template DNA by adding dNTPs from primer onward

where does pol add to?

3’ of growing strand

end of cycle 1

1 copy of dsDNA has been replicated into 2

returning to denaturing temp starts 2nd cycle

how many PCR cycles is the common standard?

25 - 30

how to resolve and assess PCR run?

gel electrophoresis

bands of desired size and intensity should be present

primer dimers

when complementary sequences within forward and reverse primers anneal to each other

non-specific bands

unwanted fragments of incorrect size are generated

how does a good PCR product look like?

strong in density and will migrate to same area

PCR troubleshooting

check primer design

condition of DNA template

were dNTPs added?

using correct DNA pol/buffer?

check thermal cycler settings

contamination?

primer design

critical component as they determine specificity of PCR

absolute specificity is ideal but not always possible

how are primers designed?

chemically manufactured on DNA synthesizer

designed to contain sequences complementary to sites flanking region to be analyzed (in template/sample)

what can you add to primers for identification?

added labels on 5’ end

all primers will be what?

ssDNA fragments, 20-30 bp

need a pair

forward primer

5’ primer

same sequence as sense strand (5’ to 3’)

binds to complementary nucleotides in antisense strand

reverse primer

3’ orimer

same sequence as antisense strand (3’ to 5’)

binds to complementary nucleotides in sense strand

things to consider for primer design

interference from secondary structure or allelic dropout

account for melting temp to assure it is similar for both primers

how to account for melting temp during primer design?

adjust by increasing length of primer or by placing primers in areas w/ more or fewer GCs

mispriming

when primer binds to unintended target

may appear as additional bands

if consuming primers/reagent in master mix, won’t get full amplification

allelic dropout

failure of some alleles to amplify during DNA analysis

DNA template

sample, 100 ng to 1 ug is used

can be genomic DNA, cDNA from RNA samples

must be free from contamination, w/o degradation

templates w/ high GC content need to be be what?

optimized for amplification

deoxyribonucleotide bases (dNTPs) info

equimolar mixture

0.1 to 0.5 mM of each nucleotide

correct concentration + free of contamination

DNA pol

thermostable enzyme

Taq pol (Tht pol also possible to use)

Taq

Thermus aquaticus

thermophilic bacterium

what factors affect DNA pol stability?

buffer, freeze-thaw, agitation

need proper buffer w/ cations

can’t have too many freeze-thaw cycles

PCR buffer

provide optimal conditions for enzyme activity - 10 mM Tris-HCl

buffer/salt ratios (KCl and (NH4)2SO4)

ions (Mg2+ from MgCl2) needed for pol activity

presence of chelators like EDTA in PCR

will lower pol activity

buffer/salt ratios (KCl and(NH₄)₂SO₄)

high salt causes longer DNA products denature more slowly

thermal cycler

heater/cooler with programmable memory to record the appropriate reaction conditions

make sure settings, temp, and time are correct

heat stability of taq pol

heat stable

has some activity at room temp - can lead to mispriming

techniques to account for taq pol mispriming

hot-start PCR

touchdown PCR

hot-start PCR

rxn mixes prepared on ice and placed in the thermal cycler after it has been prewarmed to the Tm

Taq pol isolated by monoclonal antibodies that prevent extension until they are degraded by heat in first denaturation step

touchdown PCR

PCR program starts w/ annealing temps higher than optimal target primer-binding temp

annealing temp decreased by 1°C every cycle until optimal annealing temp is reached + subsequent cycles are carried out at that ideal temp

PCR contamination

• different templates that aren’t supposed to be there

• inappropriately amplifying

• added a chelator like EDTA that shouldn’t be there

contamination checks for PCR

negative control (reagent blank)

positive control (internal control)

negative control (reagent blank)

master mix w/o DNA template

ensures primers are not annealing to nontarget sequences

positive control (internal control)

run a second set of primers and unrelated target added to rxn mix

demonstrates that rxn is working even if test sample is not amplified

controlling PCR contamination

physical separation of pre-PCR and post-PCR areas

• positive airflow w/ airlocks, dedicated disposables

UV light decontamination and 10% bleach

dUTP-UNG system

d-UTP-UNG system when controlling PCR contamination

substituting dTTP for dUTP in master mix

UNG enzyme (uracil-N-glycosylase) also added

• this enzyme will degrade any nucleic acid containing uracil, such as those PCR products from previous rxns

pcr product clean up

extraneous products and components of rxn’s master mix sometimes not completely avoided when extracting gel from DNA product

could have unwated amplicons of desired size

what is a way to obtain a clean PCR product?

resolve amplification products by gel electrophoresis, cut out band of interest, elute PCR product

process of obtaining a clean PCR product

gel containing DNA goes through a sieve in eppendorf tube, centrifuge and remove supernatant and alcohol

how are amplicons free of PCR components prepared

spin columns: DNA binds to column and rest of rxn components are rinsed away

DNA can then be eluted from column

PCR modifications

multiplex PCR (mPCR)

sequence specific primer PCR

reverse transcriptase PCR (RT-PCR)

real-time PCR/quantitative PCR (qPCR)

whole genome PCR

multiplex PCR (mPCR)

multiple amplifications that are primed using multiple primers at same time and yield multiple products

useful for diagnosis: respiratory sample can be tested for multiple pathogens and detect infection agent at same time

requires complex optimization

sequence specific primer PCR

uses primers designed to bind only a specific DNA sequence or allele

amplification occurs only if target sequence is present (PORTION of sequence)

when is sequence specific primer PCR used?

to identify SNP’s in tissue typing procedures

can identify what HLA type patient is

reverse transcriptase PCR (RT-PCR)

uses mRNA as template

mRNA → cDNA

use RT to create RNA-DNA hybrid, use RNAse to degrade RNA template + add Taq pol, giving you ss of DNA

why is the name of reverse transcriptase PCR (RT-PCR) as it is?

use special pol: reverse transcriptase from retroviruses

components of reverse transcriptase PCR (RT-PCR)

special polymerase

mRNA → cDNA (complementary DNA)

primers:

• oligo dTs that target poly-A tail of mRNA

• gene targeted-primers

• random hexamers

what is reverse transcriptase PCR (RT-PCR) used for?

eukaryotes

• gene expression analyses

detect rRNA

analyze gene regions

detect RNA viruses

real-time (quantitative) PCR (qPCR)

uses fluorescent marker or various probes

can have different targets (DNA or RNA)

what is the name of qPCR if you want to target DNA?

qPCR

what is the name of qPCR if you want to target RNA?

RT-qPCR

graphing PCR cycles using real-time (quantitative) PCR (qPCR)

# of copies = 2^N

N = # of PCR cycles

has lag, log, and stationary phase

what phase do you use in analysis of qPCR?

log (exponential)

sample with high template concentration will reach exponential phase faster (resulting in shorter lag phase)

qPCR - threshold cycle (C_T)

lag phase ends and exponential phase begins

threshold line in qPCR

level of detection or point at which rxn reaches fluorescent intensity above background levels

threshold cycle (C_T) in qPCR

# of PCR cycle at which fluorescent marker has reached above background level

this value tells how many cycles it took to detect a real signal from sample

relationship of starting concentration to C_T value in qPCR

the higher the starting concentration, the lower the C_T value

starting amount of unknown specimen can be determined

qPCR detection systems

EtBr replaced by SYBR green

artificial products also generate fluorescence

how will addition of probes in qPCR increase specificity?

by only yielding fluorescence when they hybridize to amplicon

TaqMan

uses exonuclease activity of Taq pol to generate a signal

measures fluorescent signal generated by separation of fluorescence dye and quencher in probe

what is TaqMan?

probe that is complementary to sequence in target region of PCR template

what makes up TaqMan?

fluorescent dye (R) on 5’ end

• covalently modified at 3’ end to prevent its extension

contains quencher (Q) on 3’ end

added to master mix

what does the quencher (Q) in TaqMan do?

quencher chemically shuts down fluorescent activity

how does TaqMan work?

as pol interacts w/ probe, breaks R from probe, creating detectable signal bc quencher isn’t bound

FRET Probe

Fluorescent Resonance Energy Transfer

how do FRET probes work?

uses two target specific oligonucleotides that have a donor (D) and reporter (R)

D and R oligonucleotides bind near each other and create detectable signal

how does a donor work in FRET probes?

gives away energy to fluoresce

how does a reporter work in FRET probes?

fluorescent marker

if donor not nearby, fluorescence will occur

whole genome amplification

primers used are not complementary to specific genetic regions

used to survey ALL genes or transcripts of an organism

• types of microorganisms

• screening for multiple genetic lesions