NUCLEIC ACID AMPLIFICATION 2

• PCR MODIFICATIONS

  1. Multiplex PCR
  2. Sequence-Specific PCR
  3. Reverse Transcriptase PCR
  4. Nested PCR
  5. Real-Time (Quantitative) PCR
• MULTIPLEX PCR

  • More than one primer pair can be added to a PCR
  • Multiple amplifications are primed simultaneously
  • Useful in Pathogen typing and forensic identification
  • The pattern of product sizes will be specific for a given type or individual
  • Multiplex PCR reagents and conditions require more complex optimization.
• SEQUENCE-SPECIFIC PCR

  • Used to identify single-base changes in the target DNA
  • Designing the forward or reverse primer to end with a 3ʹ base complementary to the mutant sequence
• REVERSE TRANSCRIPTASE PCR

  • The starting material is RNA
  • The conversion is accomplished through the action of Reverse Transcriptase (RT)
  • Enzyme isolated from RNA viruses
  • Product is double-stranded DNA copy or complementary DNA (cDNA)

  Primers:

  • Gene-targeted primers - highly specific for the target of interest
  • Oligo dT primers - prime cDNA synthesis only from RNA with polyA tails
  • Random Hexamers- hybridize to random sites in the target RNA
• Uses of RT-PCR:

  • Expression profiles
  • Detection of rRNA
  • Analyze gene regions interrupted by long introns
  • Detect microorganisms with RNA genomes
• Two approaches of RT-PCR

  1.One-step RT-PCR

  • Tth DNA polymerase
  • Modified amplification program

  2. Two-step RT-PCR

  • cDNA synthesis and then PCR.
• NESTED PCR

  • Modification that increases the sensitivity and specificity of the reaction.
  • Two pairs of primers are used to amplify a single target in two separate PCR runs
  • The nested PCR is the best choice in the microbial ID and 16s RNA analysis

  Nested PCR Variations:

  • Using nested primers
  • Using semi-nested second-round primers
• REAL-TIME PCR (qPCR)

  • First performed by adding ethidium bromide (EtBr) to a standard PCR
  • Tracks the accumulation of PCR products during the PCR in real time
  • Detectable fluorescence :

  - Early cycles = higher amounts of starting template

  - Later cycles = lower amounts of starting template
• ANALYSIS OF PCR PRODUCT

  Standard PCR method

  • at the end of the PCR stationary phase

  qPCR method

  • in The exponential phase of growth
• THRESHOLD CYCLE OR CT

  • PCR cycle at which sample fluorescence crosses the threshold
  • The starting amount can be determined by the cycle number at which the unknown crosses the fluorescence threshold
  • This method is applied to the quantification of DNA targets and RNA targets
• FLUORESCENT DYE

  • Dye specific to double-stranded DNA

  1.Ethidium Bromide (EtBr) - toxic

  2. SYBR green - specific and robust fluorescence; less toxic
• Mispriming and primer dimers = will also generate fluorescence
• TaqMan

  • One of the first probe-based systems for quantifying cDNA by qPCR
  • Measures the fluorescent signal generated by separation of fluorescent dye and quencher = single-stranded DNA oligonucleotide probe
  • The probe is chemically modified at its 3ʹ end so that it cannot be extended by the polymerase
  • Single-stranded DNA TaqMan probe covalently attached = fluorescent dye
  • Exonuclease activity of Taq polymerase = degrade the probe into single and oligonucleotides
  • Removal of labeled nucleotide from the vicinity of the quencher = Fluorescence
• PROBE LABELING

  1.Reporter Dye = 5’ end

  • FAM (6-carboxyfluorescein), TET (6-tetrachloroflu-orescein), HEX (6-hexachlorofluorescein), JOE (4ʹ, 5ʹ-dichloro-2ʹ, 7ʹ-dimethoxy-fluorescein), Cy3, and Cy5 (indocarbocyanine)

  2. Quencher = 3’ end

  • Fluorescent quenchers- DABCYL (4-dimethylaminophenylazobenzoic acid) or TAMRA ([56]-carboxytetramethylrhodamine)
  • Non-fluorescent quenchers- BHQ1, BHQ2 (Black Hole Quenchers), and Eclipse
• MOLECULAR BEACONS

  • Another probe-based system
  • 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
• PROBE DESIGN

  • Target-specific binding sequence of approximately 25 bases flanked by a short, approximately 5-base long stem and loop structure
  • There is a reporter fluorophore (dye) at the 5ʹ end of the oligomer and a quencher at the 3ʹ end
• SCORPION-TYPE PRIMERS

  • Intramolecular system
  • PCR product will be covalently bound to the dye
  • Target-specific primers are tailed at the 5ʹ end with a sequence complementary to part of the internal primer sequence, a quencher, a stem-loop structure, and a 5ʹ fluorophore
• Fluorescent Resonance Energy Transfer (FRET)

  • Another frequently used system
  • Utilizes two specific probes, one with a 3ʹ fluorophore (acceptor) and the other with a 5ʹ catalyst for the fluorescence (donor)
  • Donor–acceptor pairs:

  - fluorescein–rhodamine

  - fluorescein–(2 aminopurine)

  - fluorescein–Cy5
• Transcription-Based Amplification Systems (TAS)

  • Developed by Kwoh and colleagues in 1989
  • A DNA copy is synthesized from the target RNA, and then transcription of the DNA produces millions of copies of RNA
  • Commercial name variations:

  - Transcription-mediated amplification (TMA)

  - Nucleic acid sequence–based amplification (NASBA)

  - Self-sustaining sequence replication (3SR)
• Process of TAS

  1.A primer carrying the binding site for RNA polymerase is added to a sample of target RNA

  2. Primer annealing and RT makes a DNA copy of the target RNA

  3. Denaturation of DNA-RNA hybrid (Heat/RNase)

  4. A second primer binds to the cDNA and RT producing double-stranded DNA

  5. RNA polymerase (T7) transcribes the cDNA producing hundreds to thousands of copies of RNA

  6. transcribed RNA = SERVES AS TARGET RNA
• Genomic Amplification Methods

  • Amplify all regions of input DNA, or whole genomes
  • Replication initiates at random sites
  • Uses:

  - Typing of microorganisms

  - Screening for particular genetic lesions from limiting samples
• Approaches to Genomic Amplification Methods

  1.Whole-Genome Amplification

  • WGA can be performed using degenerative primers that prime random synthesis throughout the target genome or by nick translation

  2. Emulsion PCR

  • Emulsion PCR is designed to simultaneously amplify thousands of specific templates in a single reaction, producing a set of specific products, or library
• Approaches to Genomic Amplification Methods

  3. Surface Amplification (Bridge PCR)

  • Isothermal and genomic PCR method used in high-throughput sequencing technologies

  4. Arbitrarily Primed PCR

  • Also known as randomly amplified polymorphic DNA random amplification of polymorphic DNA (RAPD), short primers (10 to 15 bases) with random sequences are used to amplify arbitrary regions in genomic DNA under low-stringency
• PROBE AMPLIFICATION

  • Synthetic probes that are specific to the target sequences bind to the target
  • Probes = Amplified
  • Three major procedures involve the amplification of probe sequences:

  - Ligase chain reaction (LCR)

  - Strand displacement amplification (SDA)

  - Qβ replicase.
• Ligase Chain Reaction (LCR)

  • Amplifying synthetic primers/ probes complementary to target nucleic acid
  • The primers are bound immediately adjacent to each other
  • DNA ligase was used in LCR to ligate the adjacent primers together
  • Ligated primers = Template for annealing and ligation of additional primers
  • LCR was used to detect point mutations in a target sequence
• Ligase Chain Reaction (LCR)

  1.In LCR the reaction was heated to denature the template

  2. Primers annealed if the complementary sequence was present

  3. Thermo-stable ligase joined the two primers

  • Very sensitive, even 1-bp mismatch at the ligation point prevented ligation of the primers
• Strand Displacement Amplification (SDA)

  • Isothermal amplification process
  • Major amplification products are the PROBES
  • Widely applied to detection of M. tuberculosis

  Two stages of SDA process:

  1. TARGET GENERATION

  2. EXPONENTIAL PROBE AMPLIFICATION PHASE
• First Stage (Target Generation)

  1.Denaturation (950C)

  2. Two primers bind close to each other (inner and outer) = 5’ tail of inner primer has HincII restriction site

  3. DNA polymerase = Primer extension by incorporating modified nucleotide 2ʹ-deoxyadenosine 5ʹ-O-(1-thiotriphosphate) or dATPaS

  4. Displacement of extension products of inner primers by outer primers

  5. Second set of outer and inner primers binds to the displaced inner primer products

  6. Pol extends the complementary primers = Double stranded products
• Second Stage

  1.RE is added to the double-stranded probe DNA

  • only one strand of the probe will be cut due to the dATPαS

  2. Nick formation

  3. Extension by Polymerase enzyme

  4. Formation of ss-probe = regeneration of the restriction site (new template)

  • The reaction cycles by the strands are nicking and extension, without requirement for heat denaturation of the double-stranded DNA template (520C without temperature cycling)
• Qβ Replicase

  • Method for amplifying probes that have specificity for a target sequence
  • Qβ replicase is an RNA-dependent RNA polymerase from the bacteriophage Qβ
  • The target nucleic acid in this assay can be either DNA or RNA
  • It was used primarily to amplify the nucleic acid associated with infectious organisms, particularly mycobacteria, Chlamydia, HIV, and CMV.
• SIGNAL AMPLIFICATION

  • 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 clinical sample

  Several signal amplification methods are available commercially:

  1.Branched DNA Amplification

  2. Hybrid Capture Assays

  3. Cleavage-Based Amplification

  4. Cycling Probe
• Branched DNA Amplification (bDNA)

  • Short oligomer probes are used to capture a single target nucleic acid molecule
  • Additional extender probes bind to the target nucleic acid and then to multiple reporter molecules, loading the target nucleic acid with signal
• Hybrid Capture Assays

  • Target DNA released from cells is bound to single-stranded RNA probes
  • RNA:DNA hybrid = hybrid-specific immobilized antibodies
  • Measured by chemiluminescence
  • Primarily for the detection and molecular characterization of human papillomavirus (HPV) in genitourinary specimen
  • They are also used for the detection of hepatitis B virus and CMV
• Cleavage-Based Amplification

  • Detects target nucleic acids by using a series of overlapping probes that bind to the target DNA Cleavase
  • Bacterial enzyme that recognizes overlapping sequences of DNA
  • Cuts (cleaves) in the overlapping region
  • Applied in V laiden mutation detection, infectious diseases, such as hepatitis C virus (HCV) and HPV genotyping
• Cycling Probe

  • Target sequences are detected using a synthetic probe
  • Sequences of DNA and RNA arranged in a DNA– RNA–DNA sandwich sequence
  • After the probe binds to the target nucleic acid , Rnase H cleaves the RNA from the middle of the probe
  • The amount of fluorescence from the reporter dye (produced when the target is present) is measured as an indication of the presence of target molecules