Chapter 7: Nucleic Acid Amplification Techniques

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

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

3. Target Amplification Methods • PCR – • 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

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

5. TARGET AMPLIFICATION TECHNIQUES • 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

6. Kary Mullis and the Nobel Prize: The Basics • 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)

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

8. THREE STEPS OF PCR • Denaturation of target (template) • Usually 95oC • Annealing of primers • 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

9. 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 AMPLIFICATION BY PCR

10. PCR: First 4 Cycles

11. PCR: Completed Amplification Cycle

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

13. Features of Primers • Types of primers • Random • Specific • Primer length • Annealing temperature • Specificity • Nucleotide composition

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

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

16. ASSUMPTIONS • Product produced is product desired • 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 • Multigene families and pseudogenes

17. Thermostable DNA Polymerase: Yellowstone National Park

18. Alvin Submersible for Exploration of Deep Sea Vents

19. Thermostable Polymerases

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

21. A Standard PCR Reaction Mix 0.25 mM each primer 0.2 mM each dATP, dCTP, dGTP, dTTP 50 mM KCl 10 mM Tris, pH 8.4 1.5 mM MgCl2 2.5 units polymerase 102 - 105 copies of template 50 ml reaction volume

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

23. Combinations Of Cycle Temperatures

24. Thermostable Polymerases • Taq:Thermus aquaticus (most commonly used) • Sequenase: T. aquaticus YT-1 • 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) • Fused to DNA binding protein for processivity

25. Amplification Reaction • 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.

26. Automation of PCR • 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.

27. Avoiding Misprimes • 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. • Platinum Taq • AmpliTaq Gold • HotStarTaq

28. Primer Design • http://biotools.umassmed.edu/bioapps/primer3_www.cgi • http://arbl.cvmbs.colostate.edu/molkit/rtranslate/index.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

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

30. Contamination Control • 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.

31. Contamination of PCR Reactions • 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.

32. Pre-PCR Post-PCR Contamination Control • Physical separation • Air-locks, positive air flow • PCR hoods with UV • dUTP + 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)

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

34. Interpretation of the PCR Results • 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.

35. Molecular Marker Negative Control Positive Control Blank Reaction Patient 1 Patient 2 Patient 3 Patient 4 104 bp Diagnostic PCR AmplificationFrom Patient Samples

36. EBV b-Actin DNA Marker Specimen 1 Specimen 1 Specimen 2 Specimen 2 Specimen 1 Specimen 2 Negative Negative Positive Positive Blank Diagnostic PCR AmplificationFrom Patient Samples

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

38. PCR Modifications • 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

39. Automated PCR and Detection • The COBAS Amplicor Analyzer • 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 (qPCR) • Products are detected by fluorescence during the PCR reaction

40. Real-Time or Quantitative PCR (qPCR) • 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.

41. Quantitative PCR (qPCR) • 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.

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

43. qPCR Detection Systems • DNA-specific dyes bind and fluoresce double-stranded DNA nonspecifically. • Hybridization probes only bind and fluoresce the intended PCR product. • Primer-incorporated probes label the PCR product.

44. Sample Threshold Baseline No template

45. GEL ANALYSIS VS FLUORESCENCE

46. Threshold fluorescence level Threshold cycles for each sample Quantitative PCR (qPCR) • 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).

47. qPCR Detection Systems • DNA-specific dyes • Ethidium bromide • SyBr green • Hybridization probes • Cleavage-based (TaqMan) • Displaceable (Molecular Beacons, FRET) • Primer-incorporated probes

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

49. qPCR: SyBr Green • Binds minor groove of double- stranded DNA. • Product can be further tested in a post-amplification melt curvein which sequences have characteristic melting temperatures.

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