Nucleic Acid Amplification

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1 Nucleic Acid Amplification Chapter 7 Objectives Describe the principle of amplification by polymerase chain reaction (PCR). Discuss how products are detected. Describe examples of modifications that have been developed for PCR. Differentiate between target amplification and signal amplification. Compare and contrast between the following in vitro assays for amplifying nucleic acids: PCR, branched DNA, ligase chain reaction, transcription mediated amplification, and hybrid capture. 1

2 Polymerase Chain Reaction Primer directed in vitro enzymatic reaction for the production of a specific DNA fragment. Polymerase Chain Reaction 2

3 PCR Primers Primers are single stranded bp 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. 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). 3

4 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 mm MgCl units polymerase copies of template 50 ul reaction volume Amplification Reaction Amplification takes place as the reaction mix is subjected to an amplification program. The amplification program consists of a series of 20 to 50 PCR cycles. 4

5 PCR Cycle (Three Step) Denaturation o C, 20 seconds Annealing o C, 20 seconds Extension o C, 30 seconds PCR Cycle 1, Step 1: Denaturation 5

6 PCR Cycle 1, Step 2: Annealing PCR Cycle 1, Step 3: Extension 6

7 PCR Cycle 2 PCR Cycle 3 7

8 PCR Product = Amplicon The number of amplicons = 2 N where N = the number of cycles. Components of PCR Primers mispriming, primer dimers, tailed primers. DNA Template The best templates are in good condition, free of contaminating proteins, and without nicks or breaks that can stop DNA synthesis or cause misincorporation of nucleotide bases. Templates with high GC content and secondary structure may prove more difficult to optimizefor amplification. Deoxyribonucleotide Bases An equimolar mixture of the four deoxynucleotide triphosphates (dntps) is added to the synthesis reaction in concentrations sufficient to support the exponential increase of copies of the template. DNA Polymerase PCR Buffer Potassium chloride (20 to 1 00 mm), ammonium sulfate (1 5 to 30 mm), or other sources of monovalent cations are important buffer components. These salts affect the denaturing and annealing temperatures of the DNA and the enzyme activity. An increase in salt concentration makes longer DNA products denature more slowly than shorter DNA products during the amplification process, so shorter molecules will be amplified preferentially. Thermal Cyclers Controls for PCR A negative control without DNA (also called a contamination control or reagent blank) ensures that the reaction mix is not contaminated with template DNA or amplified products from a previous run. 8

9 Primer Design Avoid interstrand homologies. Avoid intrastrand homologies. T m of forward primer = T m of reverse primer. G/C content of 20% to 80%; avoid longer than GGGG. Product size ( bp) Target specificity 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 can be used in reversetranscriptase PCR Tli: Thermococcus litoralis Carboysothermus hydrenoformans (RT PCR) P. kodakaraensis (Thermococcus) (rapid synthesis) Pfu: Pyrococcus furiosus (fidelity) Fused to DNA binding protein for processivity 9

10 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. 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 nonspecific hybridization of primers. Primer dimers may occur due to hybridization of primers to each other. 10

Detection of PCR Product Molecular weight markers Reagent blank PCR product Misprime Primer dimers Contamination Control Any molecule of DNA containing the intended target sequence is a potential

11 Detection of PCR Product Molecular weight markers Reagent blank PCR product Misprime Primer dimers 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. 11

12 Contamination Control Physical separation Air locks, positive air flow Pre-PCR Post-PCR PCR hoods with UV Ultraviolet (uv) light has been used to decontaminate and maintain pre PCR areas. Ultraviolet light catalyzes single and double strand breaks in the DNA that will then interfere with replication. Psoralen (add to the amplification products) + uv (depends on uv wavelength and distance to surface) Psoralens intercalate between the bases of double stranded DNA, and in the presence of long wave uv light they covalently attach to the thymidines, uracils, and cytidines in the DNA chain. The bulky adducts of the psoralens prevent denaturation and amplification of the treated DNA. Contamination Control Chemical control dutp + uracil N glycosylase (added to the PCR reaction) which involves substitution of dutp for dttp in the PCR reagent master mix, resulting in incorporation of dutp instead of dttp into the PCR product. At the beginning of each PCR, the enzyme uracil N glycosylase (UNG) is added to the reaction mix. This enzyme will degrade any nucleic acid containing uracil, such as contaminating PCR product from previous reactions. A short incubation period is added to the beginning of the PCR amplification program, usually at 50 C for 2 to 15 minutes to allow the UNG enzyme to function. The initial denaturation step in the PCR cycle will degrade the UNG before synthesis of the new products 10% bleach (most effective for surface decontamination) 12

13 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 use a wax barrier A bead of wax is placed in each reaction tube containing all components of the reaction mix except enzyme and template. The tubes are heated to 1 00 C to melt the wax and then cooled to room temperature. The melted wax will float to the top of the reaction mix and congeal into a physical barrier as it cools. The remainder of the reaction mix containing template and enzyme is then added on top of the wax barrier. use a sequestered enzyme that requires an initial heat activation. Platinum Taq, AmpliTaq Gold, HotStar Taq A method called touchdown PCR is a modification of the PCR program that is also used to enhance amplification of the desired PCR product. In this method, the PCR program begins with annealing temperatures higher than the optimal target primer binding temperature. The annealing temperature is decreased by 1 C every cycle or every other cycle until the optimal annealing temperature is reached. Subsequent cycles are carried out at the optimal temperature. Any difference in the annealing temperature will translate into an exponential advantage for correct versus mismatched primer binding 13

14 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 Addition of shrimp alkaline phosphatase (SAP) in combination with exonuclease I (ExoI) is an enzymatic method for removing nucleotides and primers from PCR products prior to sequencing or mutational analyses. During a 1 5 minute incubation at 37 C, SAP dephosphorylates nucleotides, and ExoI degrades primers. The enzymes must then be removed by extraction or inactivated by heating at 80 C for 1 5 minutes. PCR Applications Structural analysis DNA typing Disease detection Cloning Mutation analysis Detection of gene expression Mapping Site directed mutagenesis Sequencing 14

15 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 Multiplex PCR More than one primer pair can be added to a PCR so that multiple amplifications are primed simultaneously, resulting in the formation of multiple products. Multiplex PCR is especially useful in typing or identification analyses. Individual organisms, from viruses to humans, can be identified or typed by observing a set of several PCR products at once. Can be used to test for the presence of more than one respiratory virus. Organisms that cause sexually transmitted diseases can be targeted using multiplex PCR and one genital swab. In a slightly different approach to testing for multiple targets, one set of primers can detect an infectious organism and a second set can then detect the presence of a gene that makes that organism resistant to a particular antimicrobial agent. 15

16 Sequence Specific PCR The strict requirement for complementarity of the 3 end of primers can be used to identify single base changes in the target DNA. By designing the forward or reverse primer to end with a 3' base complementary to the mutant sequence, the presence of the base change is detected by successful amplification. Reverse Transcriptase PCR Convert RNA to double stranded DNA Reverse transcriptase copies the RNA single strand into a RNA:DNA hybrid strand and then uses a hairpin formation on the end of the newly synthesized DNA strand to prime synthesis of the complementary DNA strand, replacing the original RNA in the hybrid. The resulting double stranded DNA is called cdna, for copy or complementary DNA Reverse transcriptase requires priming oligo dt primers or random hexamers. RT PCR is used to measure RNA expression profiles, to detect rrna, to analyze gene regions interrupted by long introns, and to detect microorganisms with RNA genomes. 16

17 Nested PCR The low level of target and the presence of interfering sequences can prevent a regular PCR from working with the reliability required for clinical applications. Nested PCR is a modification that increases the sensitivity and specificity of the reaction. In nested PCR, two pairs of primers are used to amplify a single target in two separate PCR runs. The second pair of primers, designed to bind slightly inside of the binding sites of the first pair, will amplify the product of the first PCR in a second round of amplification. The second amplification will specifically increase the amount of the intended product. In semi nested PCR, one of the second round primers is the same as the first round primer Automated PCR and Detection The COBAS Amplicor Analyzer Samples are amplified and products detected automatically after the PCR reaction. Analyzer is 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. 17

18 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. 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. 18

19 Quantitative PCR (qpcr) A threshold level of fluorescence is determined based on signal and background. Input is inversely proportional to this threshold cycle (cycle at which fluorescence crosses the threshold fluorescence level). qpcr Threshold Cycle (C T ) Threshold fluorescence level Threshold Cycles for Each Sample 19

20 qpcr Detection Systems DNA specific dyes Ethidium bromide SyBrgreen Hybridization probes Cleavage based (TaqMan) Displaceable (Molecular Beacons, FRET) Primer incorporated probes qpcr Detection Systems 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. 20

21 Denaturation PCR qpcr: SyBrGreen Binds minor groove of doublestranded DNA. Product can be further tested in a postamplification melt curve in which sequences have characteristic melting temperatures. df/dt (T m ) Temperature Probe qpcr: TaqMan R Q R = reporter dye Q = quencher R Q 21

22 Molecular Beacons measures the accumulation of product at the annealing step in the PCR cycle. The signal from Molecular Beacons is detectable only when the probes are bound to the template before displacement by the polymerase. the probe is chemically modified so that it is not degraded during the extension step a specific binding sequence of approximately 25 bases flanked by a short, approximately 5 base long inverted repeat that will form a stem and loop structure when the probe is not bound to the template. There is a reporter fluorophor (dye) at the 5' end of the oligomer and a quencher at the 3' end. Until specific product is present, the probe will form a hairpin structure that brings the fluorophore in proximity with the quencher. Fluorescence will occur onbinding of the probe to denatured template during the annealing step qpcr: Molecular Beacons R Q R Q R Q 22

23 Scorpion type primers are a variation of Molecular Beacons. In contrast to free labeled probes, the PCR product will be covalently bound to the dye. In this system, target specific primers are tailed at the 5' end with a sequence complementary to part of the internal primer sequence, a quencher, a stem loop structure, and a 5' fluorophore. The fluorophore and the quencher are positioned so that they are juxtaposed when the hairpin in the primer is intact. After polymerization, the secondary structure of the primer is overcome by hybridization of the primer sequence with the target sequence, removing the fluorophore from the quencher. qpcr: FRET Probes Examples of frequently used donor acceptor pairs are fluoresceinrhodamine, fluorescein (2 aminopurine), and fluorescein Cy5. When the donor and acceptor are brought within 1 to1 0 nm (1 to 5 bases) through specific DNA binding, excitation energy is transferred from the donor to the acceptor. The acceptor then loses the energy in the form of heat or fluorescence emission called sensitized emission. As with the molecular beacons, the more template available for binding of the probes, the more fluorescence will be generated. FRET probes are used in the medical laboratory for viral detection and quantitation and for amplification and detection of genetic diseases. 23

specialized software PCR reaction takes place in optically clear

24 D qpcr: FRET Probes D R R = reporter dye D = donor R D R D R qpcr Detection Systems Thermal cyclers with fluorescent detection and specialized software PCR reaction takes place in optically clear plates, tubes, or capillaries. Cepheid Smart Cycler Roche LightCycler 24

25 PCR Advantages Specific Simple, rapid, relatively inexpensive Amplifies from low quantities Works on damaged DNA Sensitive Flexible PCR Limitations Contamination risk Primer complexities Primer binding site complexities Amplifies rare species Detection methods 25

26 Other Amplification Methods Ligase chain reaction (LCR) Branched DNA (bdna) Hybrid capture (HC) Transcription based amplification systems Transcription mediated amplification, selfsustaining sequence replication, nucleic acid sequence based amplification (TMA, 3SR, NASBA) Transcription Mediated Amplification (TMA) Isothermal Target amplification as RNA cdna is made from RNA target adding RNA polymerase promoter. RNA is synthesized from the cdna template and can serve as a source of new cdna. M. tuberculosis, C. trachomatis, HIV, CMV 26

27 Transcription Mediated Amplification (TMA) Priming, annealing, RT Target RNA RNase H activity Second-strand synthesis RT DNA Pol RNA Pol Probe binding and amplicon detection Advantages to use TAS over PCR and other amplification procedures. First, in contrast to PCR and the ligase chain reaction (LCR, discussed below), TAS is an isothermal process, negating the requirement for thermal cycling to drive the reactions. Second, targeting RNA allows for the direct detection of RNA viruses, for example, hepatitis C virus and human immunodeficiency virus. Even targeting the RNA of organisms with DNA genomes, such as Mycobacterium tuberculosis, is more sensitive than targeting the DNA, because each bacterium, for example, has multiple copies of RNA, whereas it has only one copy of DNA 27

28 Arbitrarily Primed PCR In arbitrarily primed PCR, randomly amplified polymorphic DNA or random amplification of polymorphic DNA (RAPD), short primers (1 0 to 15 bases) with random sequences are used to amplify arbitrary regions in genomic DNA under low stringency conditions. With this method, PCR products are generated without knowing the sequence of the target or targeting a specific gene. obtain multiple products depending how many times a short sequence appears in the genome. Arbitrarily primed PCR has been used primarily in the epidemiological typing of microorganisms. Similar band patterns obtained from performing PCR with the same arbitrary primers indicate that two organisms are the same or similar Whole Genome Amplification Whole genome amplification (WGA) methods are designed to survey all genes or transcripts of an organism for purposes of typing of microorganisms or screening for particular genetic lesions. The primers used are not complementary to specific genetic regions, rather, replication initiates at random sites throughout the input nucleic acid WGA can be performed using degenerative primers that prime random synthesis throughout the target genome or by nick translation. Degenerative primers are synthetic single stranded DNA sequences that vary with some frequency at each position where N = A T G or C. WGA has multiple applications, including comparative genomic arrays, detection of single nucleotide polymorphisms, and analysis of minimal starting material, such as DNA in plasma,66 single cell analysis, and ancient DNA samples. 28

29 Probe Amplification In probe amplification procedures, the number of target nucleic acid sequences in a sample is not changed. Synthetic probes that are specific to the target sequences bind to the target where the probes themselves are amplified. Three major procedures that involve the amplification of probe sequences: ligase chain reaction (LCR), strand displacement amplification (SDA), and Qβ replicase. 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 LCR can be used to detect point mutations in a target sequence 29

30 Ligase Chain Reaction Template...GTACTCTAGCT... A G T C...CATGAGATCGA... Probes ligase Target sequences are detected by coupled and. Signal Amplification In signal amplification procedures, there is no change in the number of target or probe sequences. Large amounts of signal are bound to the target sequences that are present in the sample. Signal amplification procedures are inherently better at quantifying the amount of target sequences present in the clinicalsample. 30

31 Branched DNA (bdna) Isothermal Signal amplification A series of hybridizations attaches multiple signals to each target molecule. HBV, HCV, HIV 1 For the bdna signal amplification procedure, target nucleic acid released from the cells is denatured. The target nucleic acid binds to capture probes that are fixed to the plate well. Extender or preamplifier probes then bind to the captured target. The extender probes have sequences that are complementary to sequences in the target molecules as well as to sequences in amplifier probes. Branched DNA 31

32 Hybrid Capture Isothermal Signal amplification Immobilized DNA probes bind to RNA targets. The RNA:DNA hybrids are bound by labeled monoclonal antibodies. HPV, HBV, CMV Hybrid Capture 32

33 Cleavage based amplification Cleavage based amplification detects target nucleic acids by using a series of probes that bind to the target and overlap. Cleavase is a bacterial enzyme that recognizes overlapping sequences of DNA and makes a cut (cleaves) in the overlapping piece. In vivo, this activity is most likely important in repairing DNA. This method is the basis of the Invader system. Applications for this form of amplification include DNA polymorphisms, such as factor V Leiden mutation detection, and infectious diseases, such as HCV and HPV genotyping. 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. 33

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

AMPLIFICATION BY PCR. Target. 1. Denature. 2. Anneal primers. 3. Extend primers. Two copies of target. 1. Denature AMPLIFICATION BY PCR Target 5 3 2. Anneal primers 3 5 1. Denature Two copies of target 3. Extend primers 1. Denature 2. Anneal primers 3. Extend primers Four copies of target PCR: First 4 Cycles PCR: Completed