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. Cary 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. 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. 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. Diagnostic PCR AmplificationFrom Patient Samples

36. 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.

45. GEL ANALYSIS VS FLUORESCENCE In conventional PCR, quantification either requires multiple samples or aliquots must be taken from a single sample at certain intervals. The amplification product then has to be detected by gel electrophoresis and ethidium bromide staining or Southern blotting. These procedures have several disadvantages. Preparation of multiple samples is expensive, withdrawal of aliquots very often leads to contamination, agarose gel analysis lacks sensitivity and specificity and Southern blotting is very laborious. The accuracy of these techniques is very limited no matter which method is used. When working with the LightCycler instrument, these drawbacks to quantification are eliminated since this instrument is specially designed for online quantification in real-time.In conventional PCR, quantification either requires multiple samples or aliquots must be taken from a single sample at certain intervals. The amplification product then has to be detected by gel electrophoresis and ethidium bromide staining or Southern blotting. These procedures have several disadvantages. Preparation of multiple samples is expensive, withdrawal of aliquots very often leads to contamination, agarose gel analysis lacks sensitivity and specificity and Southern blotting is very laborious. The accuracy of these techniques is very limited no matter which method is used. When working with the LightCycler instrument, these drawbacks to quantification are eliminated since this instrument is specially designed for online quantification in real-time.

46. 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 DNA Detection with SYBR Green I DyeThe fluorescent dye SYBR Green I binds to the minor groove of the DNA double helix. In solution, the unbound dye exhibits very little fluorescence, however, fluorescence is greatly enhanced upon DNA-binding. Since SYBR Green I dye is very stable (only 6% of the activity is lost during 30 amplification cycles) and the LightCycler instrument's optical filter set matches the wavelengths of excitation and emission, it is the reagent of choice when measuring total DNA. The principle is outlined in the following figures. At the beginning of amplification, the reaction mixture contains the denatured DNA, the primers, and the dye. The unbound dye molecules weakly fluoresce, producing a minimal background fluorescence signal which is subtracted during computer analysis. After annealing of the primers, a few dye molecules can bind to the double strand. DNA binding results in a dramatic increase of the SYBR Green I molecules to emit light upon excitation. During elongation, more and more dye molecules bind to the newly synthesized DNA. If the reaction is monitored continuously, an increase in fluorescence is viewed in real-time. Upon denaturation of the DNA for the next heating cycle, the dye molecules are released and the fluorescence signal falls. Fluorescence measurement at the end of the elongation step of every PCR cycle is performed to monitor the increasing amount of amplified DNA. Together with a melting curve analysis performed subsequently to the PCR, the SYBR Green I format provides an excellent tool for specific product identification and quantification. DNA Detection with SYBR Green I DyeThe fluorescent dye SYBR Green I binds to the minor groove of the DNA double helix. In solution, the unbound dye exhibits very little fluorescence, however, fluorescence is greatly enhanced upon DNA-binding. Since SYBR Green I dye is very stable (only 6% of the activity is lost during 30 amplification cycles) and the LightCycler instrument's optical filter set matches the wavelengths of excitation and emission, it is the reagent of choice when measuring total DNA. The principle is outlined in the following figures. At the beginning of amplification, the reaction mixture contains the denatured DNA, the primers, and the dye. The unbound dye molecules weakly fluoresce, producing a minimal background fluorescence signal which is subtracted during computer analysis. After annealing of the primers, a few dye molecules can bind to the double strand. DNA binding results in a dramatic increase of the SYBR Green I molecules to emit light upon excitation. During elongation, more and more dye molecules bind to the newly synthesized DNA. If the reaction is monitored continuously, an increase in fluorescence is viewed in real-time. Upon denaturation of the DNA for the next heating cycle, the dye molecules are released and the fluorescence signal falls. Fluorescence measurement at the end of the elongation step of every PCR cycle is performed to monitor the increasing amount of amplified DNA. Together with a melting curve analysis performed subsequently to the PCR, the SYBR Green I format provides an excellent tool for specific product identification and quantification.

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

51. Cleavage-based Assay: TaqMan 5�-3� Exonuclease Cleavage (5' exonuclease)-based assay: Light emission from the reporter fluorophore (R) is quenched because of its proximity to the quencher (Q). Cleavage by Taq polymerase separates the reporter and quencher allowing fluorescence. Cleavage-Based Probes Cleavage-based probes are the most widely used real-time PCR probes and depend upon the 5' to 3' exonuclease activity of Taq DNA polymerase (Fig. 1A). This is also known as the TaqMan� assay. Forward and reverse primers are bound to the target DNA sequence, and a fluorescently labeled probe specific for the wild-type or mutant sequence is bound downstream of the forward primer. The probe is an oligonucleotide that has a fluorescent reporter dye at the 5' end and a quencher, typically TAMRA, attached to the 3' end. It is modified to prevent extension occurring from the 3' end. When the probe is intact, the proximity of the quencher reduces the fluorescence emitted by the reporter dye. During PCR, the forward primer is extended by the Taq DNA polymerase until it reaches the probe. At this point, the exonuclease activity of the Taq DNA polymerase displaces and cuts up the probe, releasing the reporter dye and the quencher. Once they are no longer in close proximity, the reporter dye emits fluorescence of a particular wavelength that can be detected. Cleavage (5' exonuclease)-based assay: Light emission from the reporter fluorophore (R) is quenched because of its proximity to the quencher (Q). Cleavage by Taq polymerase separates the reporter and quencher allowing fluorescence. Cleavage-Based Probes Cleavage-based probes are the most widely used real-time PCR probes and depend upon the 5' to 3' exonuclease activity of Taq DNA polymerase (Fig. 1A). This is also known as the TaqMan� assay. Forward and reverse primers are bound to the target DNA sequence, and a fluorescently labeled probe specific for the wild-type or mutant sequence is bound downstream of the forward primer. The probe is an oligonucleotide that has a fluorescent reporter dye at the 5' end and a quencher, typically TAMRA, attached to the 3' end. It is modified to prevent extension occurring from the 3' end. When the probe is intact, the proximity of the quencher reduces the fluorescence emitted by the reporter dye. During PCR, the forward primer is extended by the Taq DNA polymerase until it reaches the probe. At this point, the exonuclease activity of the Taq DNA polymerase displaces and cuts up the probe, releasing the reporter dye and the quencher. Once they are no longer in close proximity, the reporter dye emits fluorescence of a particular wavelength that can be detected.

52. Molecular Beacon Assay Molecular beacons: Light emission from the reporter fluorophore (R) is quenched because of its proximity to the quencher (Q), brought about by the self-complementary 5' and 3' ends of the molecular beacon, causing it to take up a hairpin loop structure. Thermal denaturation and annealing allows the central loop section of the molecular beacon to bind to its target sequence in the PCR amplicon. Thus, the reporter and quencher become sufficiently separated to allow fluorescence. In this system, the probe is not subject to cleavage by Taq polymerase. Molecular Beacons Molecular beacons are self complementary single-stranded oligonucleotides that form a hairpin loop structure (Fig. 1B) (12,13). They consist of a probe homologous to the target sequence, flanked by sequences that are homologous to each other. Attached to one end is a reporter dye (FAM, TAMRA, TET, or ROX) and to the other is attached a quencher, usually DABCYL. When the beacon binds to the target sequence, the quencher and reporter are moved apart, and fluorescence is emitted. Unlike in the cleavage-based assays, where fluorescence is detected during the elongation phase of PCR, with molecular beacons, fluorescence is detected during the annealing phase. Molecular beacons: Light emission from the reporter fluorophore (R) is quenched because of its proximity to the quencher (Q), brought about by the self-complementary 5' and 3' ends of the molecular beacon, causing it to take up a hairpin loop structure. Thermal denaturation and annealing allows the central loop section of the molecular beacon to bind to its target sequence in the PCR amplicon. Thus, the reporter and quencher become sufficiently separated to allow fluorescence. In this system, the probe is not subject to cleavage by Taq polymerase. Molecular Beacons Molecular beacons are self complementary single-stranded oligonucleotides that form a hairpin loop structure (Fig. 1B) (12,13). They consist of a probe homologous to the target sequence, flanked by sequences that are homologous to each other. Attached to one end is a reporter dye (FAM, TAMRA, TET, or ROX) and to the other is attached a quencher, usually DABCYL. When the beacon binds to the target sequence, the quencher and reporter are moved apart, and fluorescence is emitted. Unlike in the cleavage-based assays, where fluorescence is detected during the elongation phase of PCR, with molecular beacons, fluorescence is detected during the annealing phase.

53. FRET Probe Fluorescence resonance energy transfer (FRET) probes: The acceptor fluorophore (A) is unable to fluoresce until it is within 1�5 bp of the donor fluorophore. FRET Probes F�rster or fluorescence resonance energy transfer (FRET) probes are two separate fluorescently labeled oligonucleotides, one with a 5' donor molecule and the other with a 3' acceptor molecule attached: one is specific for target (i.e., wild-type or mutant) and the other is common (Fig. 1C). Only when these are placed within 1�5 bp of each other can energy be transferred from the donor to the acceptor, which then emits fluorescence. Fluorescence resonance energy transfer (FRET) probes: The acceptor fluorophore (A) is unable to fluoresce until it is within 1�5 bp of the donor fluorophore. FRET Probes F�rster or fluorescence resonance energy transfer (FRET) probes are two separate fluorescently labeled oligonucleotides, one with a 5' donor molecule and the other with a 3' acceptor molecule attached: one is specific for target (i.e., wild-type or mutant) and the other is common (Fig. 1C). Only when these are placed within 1�5 bp of each other can energy be transferred from the donor to the acceptor, which then emits fluorescence.

54. HYBRIDIZATION PROBE FORMAT FOR DNA DETECTION The Hybridization Probe format is used for DNA detection and quantification and provides a maximal specificity for product identification. In addition to the reaction components used for conventional PCR, two specially designed, sequence specific oligonucleotides labeled with fluorescent dyes are applied for this detection method. This allows highly specific detection of the amplification product as described below The top figure shows the three essential components for using fluorescence-labeled oligonucleotides as Hybridization Probes: two different oligonucleotides (labeled) and the amplification product. Oligo 1 carries a fluorescein label at its 3' end whereas oligo 2 carries another label (LC Red 640) at its 5' end. The sequences of the two oligonucleotides are selected such that they hybridize to the amplified DNA fragment in a head to tail arrangement. Why is this design important? When the oligonucleotides hybridize in this orientation, the two fluorescence dyes are positioned in close proximity to each other. The first dye (fluorescein) is excited by the LightCycler's LED (Light Emitting Diode) filtered light source, and emits green fluorescent light at a slightly longer wavelength (middle figure). When the two dyes are in close proximity (as shown in the lower figure), the emitted energy excites the LC Red 640 attached to the second hybridization probe that subsequently emits red fluorescent light at an even longer wavelength. This energy transfer, referred to as FRET (Fluorescence Resonance Energy Transfer) is highly dependent on the spacing between the two dye molecules. Only if the molecules are in close proximity (a distance between 1�5 nucleotides) is the energy transferred at high efficiency. Choosing the appropriate detection channel, the intensity of the light emitted by the LightCycler � Red 640 is filtered and measured by the LightCycler instrument's optics. The increasing amount of measured fluorescence is proportional to the increasing amount of DNA generated during the ongoing PCR process. Since LC Red 640 only emits a signal when both oligonucleotides are hybridized, the fluorescence measurement is performed after the annealing step. The Hybridization Probe format is used for DNA detection and quantification and provides a maximal specificity for product identification. In addition to the reaction components used for conventional PCR, two specially designed, sequence specific oligonucleotides labeled with fluorescent dyes are applied for this detection method. This allows highly specific detection of the amplification product as described below The top figure shows the three essential components for using fluorescence-labeled oligonucleotides as Hybridization Probes: two different oligonucleotides (labeled) and the amplification product. Oligo 1 carries a fluorescein label at its 3' end whereas oligo 2 carries another label (LC Red 640) at its 5' end. The sequences of the two oligonucleotides are selected such that they hybridize to the amplified DNA fragment in a head to tail arrangement. Why is this design important? When the oligonucleotides hybridize in this orientation, the two fluorescence dyes are positioned in close proximity to each other. The first dye (fluorescein) is excited by the LightCycler's LED (Light Emitting Diode) filtered light source, and emits green fluorescent light at a slightly longer wavelength (middle figure). When the two dyes are in close proximity (as shown in the lower figure), the emitted energy excites the LC Red 640 attached to the second hybridization probe that subsequently emits red fluorescent light at an even longer wavelength. This energy transfer, referred to as FRET (Fluorescence Resonance Energy Transfer) is highly dependent on the spacing between the two dye molecules. Only if the molecules are in close proximity (a distance between 1�5 nucleotides) is the energy transferred at high efficiency. Choosing the appropriate detection channel, the intensity of the light emitted by the LightCycler � Red 640 is filtered and measured by the LightCycler instrument's optics. The increasing amount of measured fluorescence is proportional to the increasing amount of DNA generated during the ongoing PCR process. Since LC Red 640 only emits a signal when both oligonucleotides are hybridized, the fluorescence measurement is performed after the annealing step.

55. qPCR Detection Systems Thermal cyclers with fluorescent detection and specialized software. PCR reaction takes place in optically clear plates, tubes, or capillaries.

56. Real Time PCR Instrumentation

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

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

59. 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

60. TRANSCRIPTION AMPLIFICATION METHODS 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 cDNA, followed by RNA synthesis via RNA polymerase Amplification involves synthesis of cDNA from RNA target with a primer containing the T7 RNA pol promoter sequence

61. Both NASBA and TMA Begin with RNA Amplification involves the synthesis of cDNA from the RNA target with a primer containing the T7 RNA pol promoter sequence. The Rnase H then degrades the initial strand of target RNA in the RNA-cDNA hybrid. The second primer then binds to the cDNA and is extended by the DNA pol activity of the RT, resulting in the formation of ds DNA containing the T7 RNA pol promoter. The RNA pol then generates multiple copies of ss, antisense RNA. These RNA product molecules reenter the cycle with subsequent formation of more ds cDNAs that can serve as templates for more RNA synthesis. A 109 fold amplification of the target RNA can be achieved in less than 2 hours by this method. The ss RNA products of TMA (Gen-Probe tests) are detected by modification of hybridization protection assay. Oligonucleotide probes are labeled with modified acridinium esters with either fast or slow chemiluminescent kinetics so that the signals from two hybridization reactions can be analyzed simultaneously in the same tube. The NASBA products (bioMerieux) are detected by hybridization with probes labeled with tris (2,2� bipyridine) ruthenium and electrochemiluminescence.Amplification involves the synthesis of cDNA from the RNA target with a primer containing the T7 RNA pol promoter sequence. The Rnase H then degrades the initial strand of target RNA in the RNA-cDNA hybrid. The second primer then binds to the cDNA and is extended by the DNA pol activity of the RT, resulting in the formation of ds DNA containing the T7 RNA pol promoter. The RNA pol then generates multiple copies of ss, antisense RNA. These RNA product molecules reenter the cycle with subsequent formation of more ds cDNAs that can serve as templates for more RNA synthesis. A 109 fold amplification of the target RNA can be achieved in less than 2 hours by this method. The ss RNA products of TMA (Gen-Probe tests) are detected by modification of hybridization protection assay. Oligonucleotide probes are labeled with modified acridinium esters with either fast or slow chemiluminescent kinetics so that the signals from two hybridization reactions can be analyzed simultaneously in the same tube. The NASBA products (bioMerieux) are detected by hybridization with probes labeled with tris (2,2� bipyridine) ruthenium and electrochemiluminescence.

62. Amplification involves the synthesis of cDNA from the RNA target with a primer containing the T7 RNA pol promoter sequence. The Rnase H then degrades the initial strand of target RNA in the RNA-cDNA hybrid. The second primer then binds to the cDNA and is extended by the DNA pol activity of the RT, resulting in the formation of ds DNA containing the T7 RNA pol promoter. The RNA pol then generates multiple copies of ss, antisense RNA. These RNA product molecules reenter the cycle with subsequent formation of more ds cDNAs that can serve as templates for more RNA synthesis. A 109 fold amplification of the target RNA can be achieved in less than 2 hours by this method. The ss RNA products of TMA (Gen-Probe tests) are detected by modification of hybridization protection assay. Oligonucleotide probes are labeled with modified acridinium esters with either fast or slow chemiluminescent kinetics so that the signals from two hybridization reactions can be analyzed simultaneously in the same tube. The NASBA products (bioMerieux) are detected by hybridization with probes labeled with tris (2,2� bipyridine) ruthenium and electrochemiluminescence.Amplification involves the synthesis of cDNA from the RNA target with a primer containing the T7 RNA pol promoter sequence. The Rnase H then degrades the initial strand of target RNA in the RNA-cDNA hybrid. The second primer then binds to the cDNA and is extended by the DNA pol activity of the RT, resulting in the formation of ds DNA containing the T7 RNA pol promoter. The RNA pol then generates multiple copies of ss, antisense RNA. These RNA product molecules reenter the cycle with subsequent formation of more ds cDNAs that can serve as templates for more RNA synthesis. A 109 fold amplification of the target RNA can be achieved in less than 2 hours by this method. The ss RNA products of TMA (Gen-Probe tests) are detected by modification of hybridization protection assay. Oligonucleotide probes are labeled with modified acridinium esters with either fast or slow chemiluminescent kinetics so that the signals from two hybridization reactions can be analyzed simultaneously in the same tube. The NASBA products (bioMerieux) are detected by hybridization with probes labeled with tris (2,2� bipyridine) ruthenium and electrochemiluminescence.

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

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

66. Ligase Chain Reaction Amplification of Genomic DNA

67. Ligase Chain Reaction Mutation Detection: Utilizing Mutant-Specific Oligonucleotide Primers

68. Strand Displacement Amplification Strand Displacement Amplification Like TMA and NASBA, SDA requires multiple enzymes (e.g., a thermostable polymerase and restriction enzyme) However, in contrast, it requires multiple primers in a specific order (four total) to amplify the target sequence and displace the copied sequence (Fig. 2). An additional difference is its use of a chemically modified deoxynucleotide base (thiolated dCTP). The amplification process uses two phases: the target generation phase and the amplification phase. In the target generation phase, an engineered primer that has a restriction enzyme site incorporated into it binds to its complementary target and initiates strand synthesis using a thermostable polymerase. A bumper primer displaces the strand generated from the primer containing the restriction enzyme site. Because the newly generated strands incorporate thiolated dCTP, they are not susceptible to restriction enzymatic digestion. A thermostable restriction enzyme introduces a single-strand nick in the double-stranded molecules. The thermostable polymerase then extends the new strand and thereby displaces the strand 3' to the nick. Ultimately, new strands that incorporate this restriction enzyme site lead to the exponential generation of target copies. Strand Displacement Amplification Like TMA and NASBA, SDA requires multiple enzymes (e.g., a thermostable polymerase and restriction enzyme) However, in contrast, it requires multiple primers in a specific order (four total) to amplify the target sequence and displace the copied sequence (Fig. 2). An additional difference is its use of a chemically modified deoxynucleotide base (thiolated dCTP). The amplification process uses two phases: the target generation phase and the amplification phase. In the target generation phase, an engineered primer that has a restriction enzyme site incorporated into it binds to its complementary target and initiates strand synthesis using a thermostable polymerase. A bumper primer displaces the strand generated from the primer containing the restriction enzyme site. Because the newly generated strands incorporate thiolated dCTP, they are not susceptible to restriction enzymatic digestion. A thermostable restriction enzyme introduces a single-strand nick in the double-stranded molecules. The thermostable polymerase then extends the new strand and thereby displaces the strand 3' to the nick. Ultimately, new strands that incorporate this restriction enzyme site lead to the exponential generation of target copies.

69. Branched DNA Detection 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,� �bDNA probes� and �bDNA amplifier probes.� Assay is based upon microtiter plate technology.

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

71. Branched DNA Detection

72. bDNA ASSAYS

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

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

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

76. Web Sites of Interest http://www.genscript.com/custom_service.html?&gs_cust=391826&gs_camp=316 http://www.bio.davidson.edu/courses/genomics/chip/chip.html

77. Summary 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.