2/4/2016

Integrated DNA Technologies

Basics of PCR

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Learning outcomes

Following this training, the trainee should be able to:

• Understand the purpose of PCR

• Explain briefly how the PCR mechanism works

• Understand forward and reverse primer design

• Understand important terminologies • Melting temp• Annealing temp • Homodimer• Heterodimer • Hairpin

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Components of a nucleotide

OC

C

C C

C

OH HDeoxyribose

BASE

Nucleoside

OO

O

O

P

Nucleotide

Base structures from www.wikipedia.org

Nucleic acid structure

• Nucleic acid—string of multiple nucleotides

-DNA

-RNA

3′ carbon

5′ carbon

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DNA: Deoxyribose nucleic acid

Synthesized using deoxy-nucleotide tri-phosphates (dNTPs)

“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”

-Watson and Crick

Polymerase Chain Reaction (PCR)

“PCR lets you pick out the piece of DNA you’re interested in, and make as much of it as you want”

–Kary Mullis, Nobel Prize winner, developer of PCR

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What goes into your PCR? PCR ingredients:

Water Provides liquid phase matrix

Buffer Creates optimal conditions (salt, pH)

DNA template Genomic DNA or cDNA

Enzyme Type II DNA polymerase (Taq)

Primers Oligonucleotides (~20–30 DNA bases)

dNTPs A, C, G, T (DNA building blocks)

MgCl2 Enzyme co-factor (stabilizes primer/target DNA)

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PCR cycle

Denature strands

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5′ 3′

3′ 5′

5′ 3′

3′ 5′

3′

3′

5′

5′

5′ 3′

5′ 3′

3′ 5′

3′ 5′

5′ 3′

5′ 3′

3′ 5′

3′ 5′

5′ 3′

5′ 3′

3′ 5′

3′ 5′

1

2

3

4

Base pairing of primers

Elongation of primers

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Phases of a PCR reaction

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PCR X 45 cyclesRun products on agarose gel Amplicon

Tempe

rature

Time

Denature DNA

95oC 

Anneal primers

X oC

72 oCExtension by Taq

Template• Can be genomic DNA (extracted from organism) or cDNA

• cDNA–Made from extracted RNA using reverse transcription (RT)

Splicing (remove introns)Polyadenylation

Reverse transcription(RNA  cDNA)

Transcription

1 2 3 4Intron                             Intron Intron IntronGenomic DNAExon                              Exon Exon Exon5′ 3′

1 2 3 4RNA 5′ 3′

1 2 3 4mRNA AAAAAAAAAAA5′ 3′

3′1 2 3 4 AAAAAAAAAAAA

5′mRNA

TTTTTTTTTTTTTT3′ 5′

1 2 3 4cDNA10

Primer sequence: Forward and reverse primer design• DNA has a sense and antisense strand5′ CAGTGTAGTAGTAGTCAGCGACGCGCGATCGATCGATCGCTAGCCGTCGATACGTCAGGTACGATCGTACACTGA 3′ SENSE3′ GTCACATCATCATCAGTCGCTGCGCGCTAGCTAGCTAGCGATCGGCAGCTATGCAGTCCATGCTAGCATGTGACT 5′ ANTI

• Working from the sense strand: 5′ CAGTGTAGTAGTAGTCAGCGACGCGCGATCGATCGATCGCTAGCCGTCGATACGTCAGGTACGATCGTACACTGA 3′

Forward primer Reverse primer location

• Forward primer 5′ CAGTGTAGTAGTAGTCAGCG 3′

• Reverse primer location 5′ GTCAGGTACGATCGTACAC 3′

• Reverse complement 3′ CAGTCCATGCTAGCATGTG 5′

• Reading 5′ end first 5′ GTGTACGATCGTACCTGAC 3′

Primers

• Most are between 18–30 bases long

• Long enough to ensure specificity, yet short enough to allow easy binding to the template

• Factors that determine good PCR primer design

• Primer sequence (GC content, specificity)

• Melting temperature (Tm)

• Complementarity (homo and hetero dimers)

• Secondary structures (hairpins)

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Why most primers are 18–24 bp Melting temperature and annealing temperature • Melting temperature (Tm)

-The temperature at which 50% of an oligonucleotide duplex is in single-strand form and 50% in double-strand form

-Primer pairs must have nearly equal Tmvalues (within 2°C).

• Annealing temperature (Ta)

-Temperature at which the primers can reattach to the target sequence after a denaturation step

-Ideally, Ta= (Tm) - 5°C

Both Tm and Ta directly rely on the length and composition of the primers used. Therefore, these parameters will vary among PCR experiments.

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5’                                                                                3’              

3’                                                                                5’ 

5’                              3’

3’                               5’

Secondary structures

• Primers should be screened to avoid the formation of secondary structures such as:

• Hairpins• Primer-dimers

• Homodimers—primer annealing to itself

• Heterodimers—forward primer binding to reverse primer

• Delta G > –9kcal/mole

• IDT’s OligoAnalyzer software provides this service for free

Hairpin

5′ 3′

Primer-dimer

Primer-dimer formation

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A mismatch in a primer sequence can significantly reduce melting temperature

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• Consider standard beta-actin primers and two samples—one with a mutation and one wild type

• The reduction in melting temperature in the PCR with the mutated sample could result in a higher Cq, which could be misinterpreted as a decrease in gene expression.

PCR applications

• Selectively amplify a specific region of DNA

• Determine an unknown sequence within amplified DNA

• Clone the PCR fragment in a vector• Can make RNA or protein

• Disease diagnosis• Is a certain gene transcript present?• Are there mutations present in a gene?

?

Variations of PCR

Hot start• Keep polymerase inactive during PCR preparation • Reduces non specific products

Multiplex• Use multiple primer sets to amplify different genes at the same time• Need to have different amplicon sizes to distinguish your products on gel

Nested

• 2 rounds of PCR, 2 sets of primers

• Increases specificity

Variations of PCR

Slowdown• Amplify through GC-rich regions • Use 7-deaza-2′-deoxyguanosine

Allele specific

• Identify small DNA variations between individuals

• Need specific primers and stringent conditions • RhPCR: LNA bases, 3′ mismatch

Site-directed mutagenesis

• Introduce mutations into specific genomic locations• Design primers that incorporate mutations

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rhPCR: RNase H-dependent PCR

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• Requires rhPCR primers and RNase H2 enzyme (both available from IDT)

• Primers contain a single RNA residue and a blocking moiety

• Blocked primers are activated by RNase H2 cleavage at the 5′ end of the RNA base

• Increases PCR specificity

• Eliminates primer-dimer formation and avoids undesired amplification of closely related sequences.

• More sensitive than allele-specific PCR for detection of single-nucleotide polymorphisms (SNPs)

• No band or faint band • Too few cycles were used• Poor primer design (specificity, concentration)• Extension time was too short• Annealing time was too short• Annealing temperature was too high/low • Missing reaction component

• Nonspecific bands and primer-dimers• Too many cycles were used • Excess primer • Extension time was too long• Calculated primer Tm was inaccurate• Mispriming

PCR troubleshooting • Smeared bands

• Too much template was added• Template contained an exonuclease

or was degraded• Calculated primer Tm was inaccurate

• Negative controls

• No template control (NTC)—allows detection of contamination of reagents

• No reverse transcriptase (RT)

• No amplification (DNA polymerase)

• Positive controls

• Exogenous—external template carrying target of interest (gBlocks® Gene Fragments, Ultramer® Oligonucleotides)

• Endogenous—native target present in experimental sample

PCR controls Disadvantages of traditional endpoint PCR

• Not quantitative

• Size-based discrimination only

• Results are not expressed as numbers

• Products are analyzed after 45 cycles

• Products have to be analyzed on agarose gels

• Non-automated Same starting amount Different starting amount

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Integrated DNA Technologies

Quantitative PCR (qPCR) (Real-time)

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Learning outcomes

Following this training, the trainee should be able to:

• Know the differences between PCR and qPCR

• Understand what “genotyping” and “gene expression” refer to

• Explain the differences between performing qPCR with probes vs. intercalating dyes

PCR vs. qPCR

• Is a particular DNA sequence in my sample?

Traditional PCR

• Is a particular DNA sequence present in my sample? If so, how much?

qPCR

Differences between traditional PCR and qPCR

DNA template+

Water, Buffer, Primers, dNTPs, Enzyme, MgCl2 (MASTERMIX)+

Method of detection

cgctacc

R Q

Probe (PrimeTime Assays)

R= reporter/fluorophore,Q=quencher

Sybr Green OR(fluorescentintercalating dye)

MEASURED EACH CYCLE

• A key difference in the setup of the reaction is the detection methodology

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Detection in qPCR

1 2

3 4

1 2

1 2 31 2

Intercalators

(Sybr Green)

Hybridization FRET

Molecular Beacon5’ Nuclease assay,

Prime Time Assay,

Taqman assay

cgctacc

Fam494/518 IAbkFQZ

• SYBR is a fluorescent dye not linked to a sequence.

• All other probes have the fluorescent dye attached to the nucleic acid

Cycling in PCR vs. qPCR

qPCR

Tem

pera

ture

Time

Denature DNA

95oC

Anneal primers

60 oC

72 oC

Extension by Taq

Run Product on Agarose Gel

Extension by TaqTe

mpe

ratu

re

Time

Denature DNA

95oC

Anneal primers

60 oC

+

Measure fluorescence

Traditional PCR

• 2-step cycling is common in qPCR because of time saving advantages

Fluorophore–quencher labeled qPCR probes

5′ 3′3′ 5′

5′ 3′ t c g a t c g a t c g a t a g g c c a t c g c g a c t c c t t g c t g c t

gctagct5′ 3′Primer/oligo

a g c t a g c t a g c t a t c c g g t a g c g c t g

3′ 5′

a g g a a c g a g ac

95C

60C

a c g a c g a

3′ 5′Primer/oligo

5′ R 3′taccgga

Probegcgc

Qatagcta taccgga gcgc

c t g a g g ag g t a g c g

Differences between traditional PCR and qPCR

PCR—gel based analysis at the end of amplification in the plateau phase

Traditional PCR detection

No. of DNA molecules 2500 500 100 20 4

RT-PCR

Threshold

Cycle No. 15 20 25 30 35

Plateau

qPCR

qPCR—fluorescent measurements during the entire amplification cycle

Cycle number reflects the number of copies of cDNA in a sample

Early cycle number = more DNALate cycle number = less DNA

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Primer and probe design criteria for PrimeTime® Assays

• Probes

• Length no longer than 30–35 bases• Tm value 4–10°C higher than primers• No runs of consecutive Gs• GC content 30–80%• No G at the 5' end

• Primers• Have equal Tm (60–62°C)• 15–30 bases in length• No runs of four or more Gs• Amplicon size 50–150 bp (max 400)

Genotyping vs. gene expression

• Gene expression experiments study differences in mRNA

• mRNA levels = amount of gene expression

• 5′ nuclease assays, SYBR

• Genotyping experiments study differences in genomic DNA

• Identifies which alleles are present

• LNA Probes, MGB Probes, rhPCR

Genomic DNA

mRNAProtein

Multiplex qPCR

• Amplification of multiple targets in a single reaction

FAM Cy5

• Uses different dye ratio combinations

• Choose fluorescent dyes dependent on your instrument dye filter set

• Beware of potential cross-talk between selected dyes

• Take time to calibrate your instrument, if testing a new dye

Dye selection for qPCR probes

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2/4/2016

Instrument compatibility chart

• All dyes are not compatible with all instruments

• Customer needs to check if the reporter dye he is using is compatible with his instrument

FAM

TET

HEX

JOE

MAX

Cy3

TYE 56

3

TAMRA

ROX

LC Red

 610

Texas R

ed

TEX 61

5

LC64

0

Cy5

TYE 66

5

ABI 7000 ● ○ ○ ● ○ x x ● x x x x x x xABI 7300 ● ○ ○ ● ○ x x ● x x x x x x xABI 7500 ● ○ ○ ● ○ ● ○ ● ● ○ ● ○ ○ ● ○ABI 7900 ● ● ○ ● ○ x x x x x x x x x xABI StepOne ● ○ ○ ● ○ x x x x x x x x x xABI StepOne Plus ● ○ ○ ● ○ x x ● x x x x x x xBioRad CFX384 ● ○ ● ○ ○ ○ ○ ○ ○ ○ ● ○ ○ ● ○BioRad CFX96 ● ○ ● ○ ○ ○ ○ ○ ○ ○ ● ○ ○ ● ○BioRad iCycler ● ○ ● ○ ○ ○ ○ ○ ○ ○ ● ○ ○ ● ○BioRad MiniOpticon ● ○ ● ○ ○ x x x x x x x x x xBioRad MyIQ2 ● ○ ● ○ ○ x x x x x x x x x xBioRad MyIQ5 ● ○ ● ○ ○ ○ ○ ● ○ ○ ● ○ ○ ● ○Roche LC480 ● ○ ● ○ ○ ○ ○ ○ ○ ● ○ ○ ● ● ○Stratagene Mx3000P ● ○ ● ○ ○ ● ○ ○ ○ ○ ● ○ ○ ○ ○Stratagene Mx3005P ● ○ ● ○ ○ ● ○ ○ ○ ○ ● ○ ○ ● ○

● supplier provided or recommended reporter dyes○ instrument capable dyes, but may require calibrationx instrument incapable of supporting

Instrument Compatibility with Reporter Dyes

Double-quenched probes: Next generation quenching technology

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Dyes Internal quencher 3′ quencherZEN™ Double-Quenched Probe: FAM, TET™, HEX™, MAX™, JOE ZEN™ IowaBlack® FQ

TAO™ Double-Quenched Probe: Cy® 5 TAO™ IowaBlack® RQ

PrimerQuest

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Enter RefSeq #

Upload sequence file for multiple entries

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OligoAnalyzer 3.1

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