Linear-After-The-Exponential · 2007-11-20 · “Although attractive in theory, asymmetric PCR is quite difficult to perform since the technique requires much optimization for each

LATE-PCR

Linear-After-The-Exponential

A Patented Invention of the Laboratoryof

Human Genetics and Reproductive Biology

Lab. Director: Lawrence J. Wangh, Ph.D. Department of Biology, Brandeis University, Waltham MA

2nd Nucleic Acid Quantification Meeting, London 2003

Laboratory Co-Inventors

Cristina Hartshorn

Kenneth Pierce

John Rice

J. Aquiles Sanchez

Lawrence J. Wangh

Problem: Small Sample Size

Pre-Implantation Genetic Diagnosis

The Starting Point:

Tumor Cancer Diagnosis

Problem: Tissue Heterogeneity

Additional Challenges of Clinical Importance

Solutions to General Problems in In Vitro Diagnostics:

• Improved Sensitivity of Detection

• Increased Accuracy of Detection

• Increased Reliability of Detection

• Simpler Methods and Instrumentation

• Simultaneous Detection of Multiple Targets

• Broader Range of Applications

• Construction of Automated – Integrated Systems

• Reduce Time and Cost of Detection

The LATE-PCR Platform Technologies

DNA Sequencing

Genotyping

Single-Stranded

Probes

Applications

LATE PCR

RT cDNA

Reaction Methods

QuantiLyse

PurAmp

Sample Preparation

Product Recovery

Product Analysis

Low-TmProbes

& Analysis of

Multiple Targets

Hybridization & Melting of DNA Strands is Temperature-Dependent

Tem

pera

ture

Tm, the Melting Temperature,

Hybridization 50% of the Time

Symmetric (conventional) PCRT

empe

ratu

re

n cycles

2n molecules

StrandDenaturation

Primer Extension

Primer Annealing

Probe Detection

Real-Time Symmetric PCRLimitations of Real-Time Symmetric PCR

Lower Sensitivity for Smaller Number of DNA Targets

10

100

103

105

104

107108

106

Lack of Reliability Among Replicate Samples

CT Value

Freeman et al., (1999) Biotechniques 26: 122-125

Symmetric PCR Generates High Concentrations of Both Strands

Primer Extension

Primer Annealing

StrandDenaturation

Tem

pera

ture

Primer Extension

StrandDenaturation

Tem

pera

ture

n cycles

Primer Annealing

Probe Detection

2n molecules

Why Does PCR Plateau? The Problem of Amplicon Strand Reannealing

Amplicon Strand Reannealing Competes With Primer and Probe Binding

Asymmetric PCR As a Solution to Amplicon Strand Reannealing

Phase I – Exponential Amplification

2n molecules

n cycles

Tem

pera

ture

Primer Annealing

StrandDenaturation

Primer Extension

Probe Detection

n molecules

n cycles

Tem

pera

ture

Primer Annealing

StrandDenaturation

Primer Extension

Probe Detection

Asymmetric PCR As a Solution to Amplicon Strand Reannealing

Phase II – Linear Amplification

“Although attractive in theory, asymmetric PCR is quite difficult to perform since the technique requires much optimization for each specific template-primer combination…. It is (also) important to find the optimal ratio between the two primers and the optimal amount of starting material…”

From: http://autodna.apbiotech.com/handbook/seq/seqb-18.htm

The Problem With Conventional Asymmetric PCR


LATE PCR

DNA Sequencing

SNP Genotyping

Single-Stranded

Probes

Applications

DNA Sequencing

DNA Sequencing

SNP Genotyping

SNP Genotyping

Single-Stranded

Probes

Single-Stranded

Probes

Applications

RT cDNA

RT cDNA

RT cDNA

RT cDNA

QuantiLyse

PurAmp

Sample Preparation

QuantiLyseQuantiLyse

PurAmpPurAmpPurAmp

Sample Preparation

Product Recovery

Product Analysis

Low-TmProbes

& Multiplex Probing

Product RecoveryProduct

Recovery

Product Analysis

Low-TmProbes

& Multiplex Probing

Low-TmProbes

& Multiplex Probing

Reaction Methods

Symmetric PCR

CT = 39.6 ± 1.2

Asymmetric PCRCT = 45.2 ± 1.2

• Reliable

• Efficient• Sensitive (does not plateau)

LATE-PCR

CT = 39.0 ± 0.8

Sanchez et al. (2003), In Preparation for Nature Biotechnology

The Key to LATE-PCR Primer Design

Denat.

Tem

pera

ture

Anneal

Ext.

Primer Tm

Symmetric PCR

Efficient

Asymmetric PCR

Inefficient

Primer Tm is Proportional to Primer Concentration

Efficient!

LATE-PCR

Modifies Limiting Primer So That Limiting Primer Tm Is Above Excess Primer Tm

(TmL-Tm

X) ≥ 0

Sequence nM Tm (oC)

Symmetric PCR

5’-CCTTCTCTCTGCCCCCTGGT-3’ 1000 64.8

5’-GCCAGGGGTTCCACTACGTAGA-3’ 1000 64.3

ConventionalAsymmetric

PCR

5’-CCTTCTCTCTGCCCCCTGGT-3’ 25 58.9


LATE-PCR5’- GCCCTTCTCTCTGCCCCCTGGT -3’ 25 64.0


Simple Redesign of Primers Improves Amplification Efficiency and Maintains Quantitative Kinetics of Real-Time PCR

∆Tm

- 5

+ 0

+ 0


Validation of LATE-PCR Primer Design


(TmL-Tm

X) ≥ 0

Symmetric PCR

LATE-PCR

Efficient

(TmL-Tm

X) < 0

Symmetric PCR

Asymmetric PCR

Inefficient

LATE-PCR: Rational Design Allows A Broad Range of Primer Ratios


1:10(Tm

L-TmX) = +5.5 °C

1:100(Tm

L-TmX) = +2.4 °C

Asymmetric 1:100(Tm

L-TmX) = –3.6 °C

Rule 1: (TmL – Tm

X) ≥ 0

Increasing the Value of (TmL-Tm

X) also Increases Amplification Specificity

-3 0 +5 +10

100 genomes, Annealing Temp. 2oC Below TmL

Pierce et al. (2003), In Preparation for Nucleic Acid Res.

The Problem of Product Amplicon Strand Competition

Excess Primers

Target Amplicon Strands

Product Amplicon Strands

Rule 2: (TmA – TmX) ≤ 18oC

TmATmX

Benefits of LATE-PCR Primer Design

• Increased Efficiency

LATE-PCR Provides a Rational Framework for Efficient and Reliable Amplification of Single-Stranded DNA

• Improved Sensitivity (No Plateau)

• Flexible Use of Primer Ratios


DNA Sequencing

SNP Genotyping

Single-Stranded

Probes

Applications

DNA Sequencing

DNA Sequencing

SNP Genotyping

SNP Genotyping

Single-Stranded

Probes

Single-Stranded

Probes

Applications

LATE PCR

RT cDNA

Reaction Methods

LATE PCR

LATE PCR

RT cDNA

RT cDNA

RT cDNA

RT cDNA

Reaction Methods

QuantiLyse

PurAmp

Sample Preparation


PurAmpPurAmpPurAmp

Sample Preparation


Recovery

Product Analysis

Low-TmProbes

Primer Extension

StrandDenaturation

Tem

pera

ture

Symmetric PCR

Annealing

And

Detection

Symmetric PCR

LATE-PCR Uncouples Annealing and Detection

Primer Extension

StrandDenaturation

Tem

pera

ture

Symmetric PCR

Annealing

And

Detection

Symmetric PCRLATE-PCR (linear phase)


Primer Extension

StrandDenaturation

Tem

pera

ture


Annealing Detection

LATE-PCR (linear phase)

Primer Extension

StrandDenaturation

Tem

pera

ture


Annealing


Detection

Low-Tm Probes

Primer Extension

StrandDenaturation

Tem

pera

ture


Annealing


Detection

Low-Tm Probes

High-Tm Probes

Advantages of Low-Tm Probes: Increased Allele DiscriminationAdvantages of Low-Tm Probes:

Separate Temperature Window for Primer and Probe Design

High-Tm Molecular Beacon Melting Curve

Low-Tm Molecular Beacon Melting Curve

Mutant

Wild typePrimer

Probe

17.3°C

Wild type

Mutant

Probe Primer

Sanchez et al. (2003) In Preparation for Nature Biotechnology

Advantages of Low-Tm Probes:Saturating Amounts for Increased Sensitivity


-100

200

500

800

1100

11 16 21 26 31 36 41 46 51 56

Cycle NumberFl

uore

scen

ce U

nits

0.6uM Red

1.2uM Blue

2.4uM Green

4.0uM Magenta

Low-Tm ProbesHigh-Tm Probes

-50

100

250

400

550

11 16 21 26 31 36 41 46 51 56Cycle Number

Fluo

resc

ence

Uni

ts

0.6uM Red

1.2uM Blue

2.4uM Green

4.0uM Magenta

Benefits of Low-Tm Molecular Beacons

• Do not Affect Amplification Efficiency

Low-Tm Molecular Beacons Provide Versatile and Sensitive Amplicon Detection

in LATE-PCR

• Improved Sensitivity

• Increased Allele Discrimination

• Easier to Design – Not Constrained by Primer Tm

• Expands Multiplexed Amplicon Detection


DNA Sequencing

DNA Sequencing

Single-Stranded

Probes

Single-Stranded

Probes

LATE PCR

RT cDNA

Reaction Methods

LATE PCR

LATE PCR

RT cDNA

RT cDNA

RT cDNA

RT cDNA

Reaction Methods

QuantiLyse

PurAmp

Sample Preparation


PurAmpPurAmpPurAmp

Sample Preparation

Product Recovery

Product Analysis

Low-TmProbes

& Multiplex Probing


Recovery

Product Analysis

Low-TmProbes

& Multiplex Probing

Low-TmProbes

& Multiplex Probing

SNP Genotyping

Applications

An Example of LATE-PCR Assay: Cystic Fibrosis

Pierce et al. (2003), In Preparation for Molecular Human Reproduction

Symmetric PCRCycle Number

-200

200

600

1000

1400

1800

2200

30 35 40 45 50

Cycle Number

Fluo

resc

ence

Cycle Number

-200

200

600

1000

1400

1800

2200

30 35 40 45 50

Cycle Number

Fluo

resc

ence

Homozygous

Heterozygous

Mutant

LATE-PCR

-200

200

600

1000

1400

1800

2200

30 35 40 45 50 55 60

Fluo

resc

ence

Cycle Number

Homozygous

Heterozygous

Mutant


DNA Sequencing

DNA Sequencing

Single-Stranded

Probes

Single-Stranded

Probes

LATE PCR

RT cDNA

Reaction Methods

LATE PCR

LATE PCR

RT cDNA

RT cDNA

RT cDNA

RT cDNA

Reaction Methods

QuantiLyse

PurAmp

Sample Preparation


PurAmpPurAmpPurAmp

Sample Preparation

Product Recovery

Product Analysis

Low-TmProbes

& Multiplex Probing


Recovery

Product Analysis

Low-TmProbes

& Multiplex Probing

Low-TmProbes

& Multiplex Probing

Loss of Heterozygosity

Applications

-50

250

550

850

1150

1450

1750

2050

11 16 21 26 31 36 41 46 51 56 61 66

C yc le N um be r

Fluo

resc

ence

Uni

ts

The Linear Kinetics of LATE-PCR

Homozygous

Heterozygous+

-

+

+

Loss of Heterozygous and Oncogenesis

Heterozygous

+

-

Piece of a Chromosome

Lost

+

-

Simple Assay for Loss of Heterozygosity

The American Cancer Society and National Cancer Institute recommend annual colorectal cancer screening for the more than 74 million Americans over the age of 50. In reality, only a fraction of this population is being screened routinely for this disease.

Colorectal Cancer Is a Disease That Is Well Understood From a

Genomics Point of View

:

Sequencing Mutant Amplicons Would Be Informative

But Before Sequencing it is Critical to Suppress Amplification Errors

Product Evolution

Primer Extension

Primer Annealing

StrandDenaturation

Tem

pera

ture

Primer Extension

Primer Annealing

StrandDenaturation

Tem

pera

ture

n cycles

n molecules

Primer Annealing

Probe Detection

In LATE-PCR: Double-Stranded DNA Molecules Should Remain Constant During Linear Amplification

The Phenomenon of Product Evolution

No Evolution: ds-Molecules Constant + Evolution: ds-Molecules Increase

Stringent Conditions Non-Stringent Conditions

Hypotheses I to Explain Product Evolution

Denat.

Tem

pera

ture

Anneal.

Ext.

Detection

Solution: Primer Reverse Complement

Primer

Reverse Complement

Primer Mis-Priming

Of the Single-Strands

n

p53 Mutations

• found in the majority of Li-Fraumeni syndrome cases, an autosomal dominantly inherited disorder• most frequently observed somatic genetic events, occurring in ~50% of all cancers

600 nucleotides

Amplification of the p53 Exon 7-8 Amplicon

600 bp

88oC

Reverse complements

No reverse complements

LATE PCR Analysis of p53 gene from Single CellDouble Stranded Probe and Two Reverse Complements

-100

400

900

1400

1900

35 39 43 47 51 55 59 63 67

Cycle Number

Fluo

resc

ence

Uni

ts

600 bp


DNA Sequencing

Applications

SNP Genotyping

SNP Genotyping

Single-Stranded

Probes

Single-Stranded

Probes

LATE PCR

RT cDNA

Reaction Methods

LATE PCR

LATE PCR

RT cDNA

RT cDNA

RT cDNA

RT cDNA

Reaction Methods

QuantiLyse

PurAmp

Sample Preparation


PurAmpPurAmpPurAmp

Sample Preparation

Product Recovery

Product Analysis

Low-TmProbes

& Multiplex Probing


Recovery

Product Analysis

Low-TmProbes

& Multiplex Probing

Low-TmProbes

& Multiplex Probing

PCR in the presence of biotin-modified

primer

Binding of PCR products to

streptavidin-coated beads

Washing, discharge of strand with

unmodified primer, and neutralization

Annealing of bead-attached template to sequencing primer

PyrosequencingAnalysis

LATE-PCR

Add sequencing primer

The Problem

The Solution


primer


primer













LATE-PCRLATE-PCR



The Problem

The Solution

The Problem of Substrate Preparation for Pyrosequencing

Pyrosequencing


DNA Sequencing

SNP Genotyping

Single-Stranded

Probes

Applications

DNA Sequencing

DNA Sequencing

SNP Genotyping

SNP Genotyping

Single-Stranded

Probes

Single-Stranded

Probes

Applications

LATE PCR

RT cDNA

Reaction Methods

LATE PCR

LATE PCR

RT cDNA

RT cDNA

RT cDNA

RT cDNA

Reaction Methods

QuantiLyse

PurAmp

Sample Preparation


PurAmpPurAmpPurAmp

Sample Preparation

Low-TmProbes

& Multiplex Probing

Low-TmProbes

& Multiplex Probing

Product Recovery

Product Analysis

Primer Extension

Primer Annealing

StrandDenaturation

Tem

pera

ture

Primer Extension

Primer Annealing

StrandDenaturation

Tem

pera

ture

n cycles

n molecules

Primer Annealing

Probe Detection

Automated Product Recovery

PCR Reaction Chamber

Collection Chambers

The Race Track Reaction Chamber: Amplification & Product Recovery in a Closed System Format

Benefits of the RaceTrack Reaction Chamber

• Enables Simultaneous Amplification of Multiple Targets

• Eliminates Product Evolution

• Unlimited Single-Strand DNA Yield

The Race Track Reaction Chamber is a Closed Tube Solution for Multiplex

LATE-PCR Applications

• Automatable – No Operator Input Required


CHIP FORMAT

DNA Sequencing

SNP Genotyping

Single-Stranded

Probes

Applications

LATE PCR

RT cDNA

Reaction Methods

QuantiLyse

PurAmp

Sample Preparation

Product Recovery

Product Analysis

Low-TmProbes

& Multiplex Probing

Why Will Users Adopt These Technologies ?

• SLIO: Suppression of Amplification Errors in all Forms of PCR

• Quantitative End Point Assays

• Direct DNA Sequencing

• Primer Design Made More Rational

• Thermal Cycle Profiles Made More Precise and Easier to Design

• Simple One-Tube Methods for Preparation of DNA and/or RNA

• Rational Approaches to Analysis and Recovery of Multiple Targets

• Probe Design Made Easier and More Reliable

• PurAmp: Quantitative RT-PCR Using Internal DNA as Controls

Additional Advantages of LATE-PCR

• Compatible With Existing PCR Equipment

• Lower Cost (Greater Sensitivity Means Less Reagents)

• Ideal for Lab-On-A-Chip Format

LATE-PCR and Several Allied Technologies

Are Available for Licensing

From Brandeis University

Documents

Linear-After-The-Exponential · 2007-11-20 · “Although attractive in theory, asymmetric PCR is quite difficult to perform since the technique requires much optimization for each