Chapter 7 Nucleic Acid Amplification Techniques Donna C

Chapter 7: Nucleic Acid Amplification Techniques Donna C. Sullivan, Ph. D Division of Infectious Diseases University of Mississippi Medical Center

MOLECULAR AMPLIFICATION TECHNIQUES l Nucleic acid (NA) amplification methods fall into 3 categories l l l Target amplification systems Probe amplification systems Signal amplification

Target Amplification Methods l PCR – l l l l PCR using specific probes RT PCR Nested PCR-increases sensitivity, uses two sets of amplification primers, one internal to the other Multiplex PCR-two or more sets of primers specific for different targets Arbitrarily Primed PCR/Random Primer PCR NASBA - Nucleic Acid Sequence-Based Amplification TMA – Transcription Mediated Amplification SDA - Strand Displacement Amplification

Signal and Probe Amplification Methods l Signal Amplification l l l b. DNA – Branched DNA probes Hybrid Capture – Anti-DNA-RNA hybrid antibody Probe Amplification l l LCR – Ligase Chain Reaction Cleavase Invader – FEN-1 DNA polymerase (cleavase)

TARGET AMPLIFICATION TECHNIQUES l l All use enzyme-mediated processes, to synthesize copies of target nucleic acid Amplification products detected by 2 oligonucleotide primers Produce 108 -109 copies of targeted sequences Sensitive to contamination, false-positive reaction

Kary Mullis and the Nobel Prize: The Basics l l l Knew that you could expose template DNA by boiling ds DNA to produce ss DNA Knew that you could use primers to initiate DNA synthesis Knew that a cheap, commercial enzyme was available (Klenow fragment of E. coli DNA polymerase)

Cary Mullis and PCR l l Wanted a way to generate large amounts of DNA from a single copy Initially used the “ 3 graduate student” method l l l Denaturing Annealing Extending

THREE STEPS OF PCR l Denaturation of target (template) l l Annealing of primers l l l Usually 95 o. C Temperature of annealing is dependent on the G+C content May be high (no mismatch allowed) or low (allows some mismatch) stringency Extension (synthesis) of new strand

AMPLIFICATION BY PCR Target 5’ 3’ 3’ 5’ 1. Denature 2. Anneal primers 3. Extend primers Two copies of target 1. Denature 2. Anneal primers 3. Extend primers Four copies of target

PCR: First 4 Cycles

PCR: Completed Amplification Cycle

POLYMERASE CHAIN REACTION l l Primers (may be specific or random) Thermostable polymerase l l Taq pol Pfu pol Vent pol Target nucleic acid (template) l l Usually DNA Can be RNA if an extra step is added

Features of Primers l l l Types of primers l Random l Specific Primer length l Annealing temperature l Specificity Nucleotide composition

PCR Primers are single-stranded 18– 30 b DNA fragments complementary to sequences flanking the region to be amplified. l Primers determine the specificity of the PCR reaction. l The distance between the primer binding sites will determine the size of the PCR product. l

Tm l For short (14– 20 bp) oligomers: l Tm = 4° (GC) + 2° (AT)

ASSUMPTIONS l Product produced is product desired l l l There is always the possibility of mismatch and production of artifacts However, if it is the right size, its probably the right product Product is from the orthologous locus l Multigene families and pseudogenes

Thermostable DNA Polymerase: Yellowstone National Park

Alvin Submersible for Exploration of Deep Sea Vents

Thermostable Polymerases

Performing PCR Assemble a reaction mix containing all components necessary for DNA synthesis. l Subject the reaction mix to an amplification program. l Analyze the product of the PCR reaction (the amplicon). l

A Standard PCR Reaction Mix 0. 25 m. M each primer 0. 2 m. M each d. ATP, d. CTP, d. GTP, d. TTP 50 m. M KCl 10 m. M Tris, p. H 8. 4 1. 5 m. M Mg. Cl 2 2. 5 units polymerase 102 - 105 copies of template 50 ml reaction volume

PCR Cycle: Temperatures l Denaturation temperature l l l Annealing temperature l l l Reduce double stranded molecules to single stranded molecules 90– 96 o. C, 20 seconds Controls specificity of hybridization 40– 68 o. C, 20 seconds Extension temperature l l Optimized for individual polymerases 70– 75 o. C, 30 seconds

Combinations Of Cycle Temperatures

Thermostable Polymerases l l l l Taq: Thermus aquaticus (most commonly used) l Sequenase: T. aquaticus YT-1 l Restorase (Taq + repair enzyme) Tfl: T. flavus Tth: T. thermophilus HB-8 Tli: Thermococcus litoralis Carboysothermus hydrenoformans (RT-PCR) P. kodakaraensis (Thermococcus) (rapid synthesis) Pfu: Pyrococcus furiosus (fidelity) l Fused to DNA binding protein for processivity

Amplification Reaction l l Amplification takes place as the reaction mix is subjected to an amplification program. The amplification program consists of a series of 20– 50 PCR cycles.

Automation of PCR l l l PCR requires repeated temperature changes. The thermal cycler changes temperatures in a block or chamber holding the samples. Thermostable polymerases are used to withstand the repeated high denaturation temperatures.

Avoiding Misprimes l l Use proper annealing temperature. Design primers carefully. Adjust monovalent cation concentration. Use hot-start: prepare reaction mixes on ice, place in preheated cycler or use a sequestered enzyme that requires an initial heat activation. l l l Platinum Taq Ampli. Taq Gold Hot. Star. Taq

Primer Design l l l l http: //biotools. umassmed. edu/bioapps/primer 3_www. cgi http: //arbl. cvmbs. colostate. edu/molkit/rtranslate/inde x. html Avoid inter-strand homologies Avoid intra-strand homologies Tm of forward primer = Tm of reverse primer G/C content of 20– 80%; avoid longer than GGGG Product size (100– 700 bp) Target specificity

Product Cleanup l Gel elution l l l Removes all reaction components as well as misprimes and primer dimers Solid phase isolation of PCR product (e. g. , spin columns) DNA precipitation

Contamination Control l Any molecule of DNA containing the intended target sequence is a potential source of contamination. The most dangerous contaminant is PCR product from a previous reaction. Laboratories are designed to prevent exposure of pre-PCR reagents and materials to post-PCR contaminants.

Contamination of PCR Reactions l l l l Most common cause is carelessness and bad technique. Separate pre- and post-PCR facilities. Dedicated pipettes and reagents. Change gloves. Aerosol barrier pipette tips. Meticulous technique 10% bleach, acid baths, UV light Dilute extracted DNA.

Contamination Control l Physical separation l l l Air-locks, positive air flow PCR hoods with UV d. UTP + uracil-N-glycosylase (added to the PCR reaction) Psoralen + UV (depends on UV wavelength and distance to surface) 10% bleach (most effective for surface decontamination) Pre-PCR Post-PCR

Polymerase Chain Reaction Controls for PCR l Blank reaction l l l Negative control reaction l l l Controls for contamination Contains all reagents except DNA template Controls for specificity of the amplification reaction Contains all reagents and a DNA template lacking the target sequence Positive control reaction l l Controls for sensitivity Contains all reagents and a known target-containing DNA template

Interpretation of the PCR Results l l The PCR product should be of the expected size. No product should be present in the reagent blank. Misprimes may occur due to non-specific hybridization of primers. Primer dimers may occur due to hybridization of primers to each other.

104 bp Molecular Marker Patient 4 Patient 3 Patient 2 Patient 1 Positive Control Negative Control Blank Reaction Diagnostic PCR Amplification From Patient Samples

Specimen 2 Specimen 1 Positive EBV Negative DNA Marker Specimen 2 Specimen 1 Positive Negative Blank Diagnostic PCR Amplification From Patient Samples b-Actin

PCR Applications Structural analysis l DNA typing l Disease detection l Cloning l Mutation analysis l Detection of gene expression l Mapping l Site-directed mutagenesis l Sequencing l

PCR Modifications l l l l l Nested PCR Multiplex PCR Tailed primers Sequence-specific PCR Reverse-transcriptase PCR Long-range PCR Whole-genome amplification RAPD PCR (AP-PCR) Quantitative real-time PCR

Automated PCR and Detection l The COBAS Amplicor Analyzer l l l Samples are amplified and products detected automatically after the PCR reaction Used for infectious disease applications (HIV, HCV, HBV, CMV, Chlamydia, Neisseria, Mycobacterium tuberculosis) Real-time or quantitative PCR (q. PCR) l Products are detected by fluorescence during the PCR reaction

Real-Time or Quantitative PCR (q. PCR) l l l Standard PCR with an added probe or dye to generate a fluorescent signal from the product. Detection of signal in real time allows quantification of starting material. Performed in specialized thermal cyclers with fluorescent detection systems.

Quantitative PCR (q. PCR) l l l PCR product grows in an exponential fashion (doubling at each cycle). PCR signal is observed as an exponential curve with a lag phase, a log phase, a linear phase, and a stationary phase. The length of the lag phase is inversely proportional to the amount of starting material.

SEQUENCE DETECTION APPLICATIONS l End point PCR: simple +/- results l l l Real time PCR: complex results l l PCR product detection (pathogens, transgenes) Genotyping (allelic discrimination, single nucleotide polymorphisms-SNPs) Absolute quantitation Relative quantitation PCR interrogation (optimization) Hybridization analysis: probe hybridization

q. PCR Detection Systems l l l DNA-specific dyes bind and fluoresce doublestranded DNA nonspecifically. Hybridization probes only bind and fluoresce the intended PCR product. Primer-incorporated probes label the PCR product.

Sample Threshold Baseline No template

GEL ANALYSIS VS FLUORESCENCE

Quantitative PCR (q. PCR) l l A threshold level of fluorescence is determined based on signal and background. Input is inversely proportional to “threshold” cycle (cycle at which fluorescence crosses the threshold fluorescence level). Threshold fluorescence level Threshold cycles for each sample

q. PCR Detection Systems l DNA-specific dyes l l l Hybridization probes l l l Ethidium bromide Sy. Br green Cleavage-based (Taq. Man ) Displaceable (Molecular Beacons , FRET ) Primer-incorporated probes

DNA Detection: SYBR Green I Dye DENATURATION STEP: DNA + PRIMERS + DYE WEAK BACKGROUND FLUORESCENCE ANEALING STEP: DYE BINDS ds. DNA, EMITS LIGHT EXTENSION STEP: MEASURE LIGHT EMMISSION

q. PCR: Sy. Br Green § Binds minor groove of doublestranded DNA. § Product can be further tested in a post-amplification melt curve in which sequences have characteristic melting temperatures.

Real-Time PCR Labeled Probes l Cleavage-based probes l l l Molecular beacons l l l Taq. Man Assay Fluorescent reporter at 5’ end a quencher at 3’ end Hairpin loop structure Fluorescent reporter at 5’ end a quencher at 3’ end FRET probes l Fluorescence resonance energy transfer probes

Cleavage-based Assay: Taq. Man 5’-3’ Exonuclease Dual labeled Probe Cleavage of Dual labeled Probe

Molecular Beacon Assay

FRET Probe

HYBRIDIZATION PROBE FORMAT FOR DNA DETECTION DENATURATION STEP: DNA + TWO FLUORESCENT PROBES ANNEALING STEP: PROBES BIND VERY NEAR ONE ANOTHER EXTENSION STEP: ENERGY OF EXCITATION FROM ONE PROBE TRANSFERRED TO THE OTHER (FLUORESECENCE RESONANCE ENERGY TRANSFER, FRET)

q. PCR Detection Systems Thermal cyclers with fluorescent detection and specialized software. l PCR reaction takes place in optically clear plates, tubes, or capillaries. l Cepheid Smart Cycler Roche Light. Cycler

Real Time PCR Instrumentation 5700 Applied Biosystems i. Cycler Bio. Rad 7700 Applied Biosystems Light. Cycler real-time PCR Roche real-timehardware Fluor. Tracker Stratagene Fluor. Imager Molecular Dynamics

PCR Advantages Specific l Simple, rapid, relatively inexpensive l Amplifies from low quantities l Works on damaged DNA l Sensitive l Flexible l

PCR Limitations Contamination risk l Primer complexities l Primer-binding site complexities l Amplifies rare species l Detection methods l

Target Amplification Methods l PCR – l l l l PCR using specific probes RT PCR Nested PCR-increases sensitivity, uses two sets of amplification primers, one internal to the other Multiplex PCR-two or more sets of primers specific for different targets Arbitrarily Primed PCR/Random Primer PCR NASBA - Nucleic Acid Sequence-Based Amplification TMA – Transcription Mediated Amplification SDA - Strand Displacement Amplification

TRANSCRIPTION AMPLIFICATION METHODS l l Nucleic acid sequence based amplification (NASBA) and transcription mediated amplification (TMA) Both are isothermal RNA amplifications modeled after retroviral replication RNA target is reverse transcribed into c. DNA, followed by RNA synthesis via RNA polymerase Amplification involves synthesis of c. DNA from RNA target with a primer containing the T 7 RNA pol promoter sequence

Both NASBA and TMA Begin with RNA

Probe and Signal Amplification Methods l Probe Amplification l l LCR – Ligase Chain Reaction Strand Displacement Amplification Cleavase Invader – FEN-1 DNA polymerase (cleavase) Signal Amplification l l b. DNA – Branched DNA probes Hybrid Capture – Anti-DNA-RNA hybrid antibody

Ligase Chain Reaction Isothermal l Probe amplification l Probes bind immediately adjacent to one another on template. l The bound probes are ligated and become templates for the binding of more probes. l C. trachomatis, N. gonorrhoeae, sickle cell mutation l

Ligase Chain Reaction Template Probes . . . GTACTCTAGCT. . . AG T C. . . CATGAGATCGA. . . ligase Target sequences are detected by coupled and .

Ligase Chain Reaction Amplification of Genomic DNA Primer 1 Target Primer 2 Target Annealing Ligation Additional cycles of denaturation, annealing, ligation

Ligase Chain Reaction Mutation Detection: Utilizing Mutant-Specific Oligonucleotide Primers Wild-Type Sequence Mutant Sequence Annealing Ligation No DNA Products DNA Product

Strand Displacement Amplification

Branched DNA Detection l l Target nucleic acid sequences are not replicated through enzymatic amplification. Detection sensitivity is provided by amplification of the signal from the probe. Uses “capture probes, ” “b. DNA probes” and “b. DNA amplifier probes. ” Assay is based upon microtiter plate technology.

b. DNA ASSAYS l l Solid phase signal amplification system Multiple sets of synthetic oligonucleotide probes l l l Capture probes bound to well Target specific probes Amplifier molecule with 15 identical branches, each of which can bind to 3 labeled probes

Branched DNA Detection Hybridize b. DNA Probe Target: Capture Probe Hybridize b. DNA Amplifier Addition of Alkaline Phosphatase Molecules

b. DNA ASSAYS

HYBRID CAPTURE ASSAY l l Solution hybridization, antibody capture assay Chemiluminescence detection of hybrid (DNA/RNA) molecules l l l DNA is denatured Hybridized to RNA probe Captured by bound anti DNA/RNA antibodies

Hybrid Capture Assay l Release Nucleic Acids l l Clinical specimens are combined with a base solution which disrupts the virus or bacteria and releases target DNA. Hybridize RNA Probe with Target DNA l Target DNA combines with specific RNA probes creating RNA: DNA hybrids.

Hybrid Capture Assay l Capture Hybrids l l RNA: DNA hybrids are captured onto a microtiter well coated with capture antibodies specific for RNA: DNA hybrids. Label for Detection l Captured RNA: DNA hybrids are detected with multiple antibodies conjugated to alkaline phosphatase

Web Sites of Interest l l http: //www. genscript. com/custom_service. ht ml? &gs_cust=391826&gs_camp=316 http: //www. bio. davidson. edu/courses/genomi cs/chip. html

Summary l l l PCR is a method to specifically amplify target sequences in a complex mixture. The primers determine what sequences are amplified (specificity). Contamination control is important in laboratories performing PCR. Quantitative PCR offers the advantage of quantifying target. In addition to PCR, signal and probe amplification methods are available for use in the clinical laboratory.