Polymerase Chain Reaction
-amplify specific DNA sequences
-need primers, reactions mix, amplicon
Target (Template) DNA : The DNAextracted from the specimen
dNTPs (deoxyribonucleoside triphosphates-The “building blocks” added to ss of DNA by Taq polymerase to make new ds of DNA
Primers (Forward & Reverse):
2 short sequences of complementary DNA or RNA (20 base pairs)
* Primers determine the specificity of the PCR reaction
* The distance between the primer binding sites will determine of the size of the PCR product
DNA polymerase:
Enzyme in cells that use existing single strands of DNA as a template to make new double strands of DNA
Reads the template and adds nucleotides one at a time
In PCR use Taq polymerase because it does not denature at the high temperatures required in each PCR cycle
- Taq: Thermus aquaticus (most commonly used)
- Sequenase: T. aquaticus YT-1
- Restorase (Taq + repair enzyme
PCR STEPS
Denaturation 90–96oC * 20 seconds
-temperature increased to separate the DNA strands
Annealing 50-65 1-2min
Temperature decreased to allow primers to base pair to complementary DNA template
Extension 68-72 10-15 min
Polymerase extends primer to form nascent DNA strand
PCR Product = Amplicon
The number of amplicons = 2N where N = the number of cycles
Automation of PCR
The thermal cycler changes temperature in a block or chamber holding the samples
* Thermostable polymerases are used
Interpretation of PCR Results
Easiest way to visualize PCR product is by agarose gel electrophoresis
-Nucleic acids or proteins separated based on size, charge and shape by an electric charge (from – to +)
-Visualized by staining with dye (fluorescence)
-Compared to a ladder (size standard ruler) included on one lane of the gel
*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
Avoiding Mis-Primes & Primer Dimers
- Use proper annealing temperature
- Design primers carefully
- Adjust monovalent cation concentration
- Use hot-start method: prepare reaction mixes on ice, place in
preheated cycler, or use a sequestered enzyme that requires an initial heat activation
Primer Design
- Avoid inter/intra strand homologies
- Tm of forward primer = Tm of reverse primer
- G/C content of 20% to 80%; avoid longer than GGGG
- Product size (100 to 700 base pairs)
- Target specificity
Product Cleaning
- Purpose: Removes all reaction components as well as misprimes and primer dimers
- Gel elution
- Solid-phase isolation of PCR product (example, spin columns)
- DNA precipitation
Contamination Control
- Any molecule of DNA containing the intended target sequence is a
potential source of contamination - Most dangerous contaminant is the PCR product from the previous
reaction
Physical separation (air-locks, positive air flow), UV, 10% bleach
PCR Modifications:
nested primer
tailed primer
nested: product of the first amplification reaction is used as the template for the second PCR
tailed: contains at the 5’ side, the sequence of the gene you want to amplify. controls sequence ensuring the cloning into vectors
Quantitative PCR (qPCR)
-qPCR combines PCR amplification and detection into a single step.
-PCR amplification products are assayed as they accumulate rather than at the end of the procedure
-Positive result can be observed while assay is still running
-Uses a fluorescent reporter dye or a probe labelled with a fluorescent dye
-The Thermocyler used will also have a UV light source to excite the reporter & a camera to detect increasing fluorescence at each cycle of amplification
- PCR product grows in an exponential fashion (doubling at each cycle) 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
qPCR – Detection Systems
- DNA-specific dyes
- Ethidium bromide
- SYBR green
- Hybridization probes
- Cleavage-based (TaqMan)
- Displaceable (molecular beacons, FRET)
- Primer-incorporated probes
qPCR – SYBR Green
*Fluorescent dye that binds to any double strand of DNA
*less specific than Taqman
* Binds minor groove of double stranded DNA
* Product can be further tested in
a post- amplification melt curve
where sequences have
characteristic melting temperatures
qPCR – TaqMAN
- short sequence of DNA specific to a section of the target DNA.
-fluorescent dye on its 5’ end and quencher on its 3’ - closed no fluorescence
-hybridizes between the primer sites and get digested via exonuclease activity of DNA polymerase
-glows after getting digested
The amount of signal depends on the number of targets amplified
qPCR – Fluorescence Resonance Energy Transfer (FRET) Probes
They bind adjacent to one another on the target
* Allowing the donor to transfer energy to the reporter dye
* The amount of signal depends on the amount of target present
qPCR – Molecular Beacons
- Molecular beacons bind target to produce signal
- The reporter (R) and quencher (Q) are on opposite ends of the probe and held together by about 5 bp of homology when not bound to the target bound to each other like a key shape and then they become linear when binding DNA
- In the presence of target sequences, the probe binding overcomes the short-end homology, releasing the reporter from the quencher
qPCR – Scorpion Probes
- Scorpion primer-binding site is added to any target using a 5’ tailed primer in an initial PCR
reaction
-fluorescent signal is covalently attached to the PCR product - allowing subsequent analysis, for instance, by capillary electrophoresis
Point Mutations
- Point mutation involve one or few base pairs
- Do not always have a phenotypic effect
silent mutation - change in DNA sequence does not affect sequence of aa
conservative mutation - aa substitution with another aa having no impact
non conservative - aa substitution has different properties than original ie sickle cell
nonsense - change in DNA that causes a protein to terminate or end translation early
frameshift - insertion or deletion of nucleotide bases that are not multiples of 3 because a cell reads a genes code in multiples of three
Biochemical Methods to detect mutations
Immunoassay
HPLC
– Gas Chromatography
Immunoassay
* Antibodies that detect mutant or wild-type proteins
* Immunohistochemistry in tissue
HPLC
*Uses solid and liquid phases to separate particles by size, charge, or chemical characteristics
Gas Chromatography
* Samples are vaporized and
separated in a gas phase
-MALDI-TOF using ddNTPs
Nucleic Acid-Based Detection Methods :* Hybridization based
Single-Strand Conformation
Polymorphism (SSCP)
Melt-Curve Analysis
Array Technology
Single-Strand Conformation Polymorphism (SSCP)
- Single-strand conformational polymorphism (SSCP), melt curves, array technology
-one base affects folding
-strict temp requirements
Melt-Curve Analysis Based on sequence effect on Tm
* Dye specific to double-stranded DNA (SYBR green) or FRET probes will fluoresce when bound to DNA
* Denaturation of DNA to single strand will result in loss of fluorescence
* The speed of drop of fluorescence (dF) will be maximal at the Tm
* Every sequence has a characteristic Tm
* Melt-curve Tm indicates which sequence is present
Array Technology
*reverse-dot-blot methods
* used to investigate multiple genomic sites simultaneously
* Unlabeled probes are bound to substrate
* Specimen DNA is labeled and hybridized to immobilized probes
Nucleic Acid-Based Detection Methods : Sequencing-based
Sequence-Specific Primer PCR (SSPPCR)
Allelic Discrimination
Sequence-Specific Primer PCR (SSPPCR)
* PCR primer extension requires that the 3’ base of the primer is
complementary to the template
* Used to detect point mutation
* Presence or absence of product interpreted as presence or absence of the mutation
Allelic Discrimination
* Uses fluorescently labeled probes
* Quantitative PCR (qPCR) technology
* Generates “color” signal for mutant or normal sequence
* Performed on real-time PCR instruments
Nucleic Acid-Based Detection Methods : Cleavage-based
Restriction Fragment Length
Polymorphism (RFLP)
Restriction Fragment Length
Polymorphism (RFLP)
* Restriction enzyme site recognition detects presence of sequence changes
* For example, G→A change creates EcoR1 site
Heteroduplex Analysis with Single-Strand-Specific
Nucleases
- Uses nucleases that cut singles tranded bubbles in heteroduplexes
- Region of interest is amplified by PCR or transcribed
- The double-stranded (DS) product is denatured and renatured with or without added normal product
- Renatured duplexes are digested with nuclease
- Products are observed by gel
electrophoresis
DNA Sequencing Methods
- Maxam & Gilbert chemical sequencing
- Sanger chain termination sequencing
- Pyrosequencing
- Next-generation sequencing
Sanger – Chain Termination Sequencing
- A modified DNA replication reaction
- chains are terminated by dideoxynucleotides
- The 3′-OH group necessary for formation of the phosphodiester bond is missing in ddNTP’s and this will terminate extension
-* Dideoxynucleotides are added separately to each of four tubes proper ratio of dNTP:ddNTP - DNA polymerase will extend the primer until a ddNTP is encountered
- The chain will end with the incorporation of the ddNTP
- The collection of fragments is a sequencing ladder
- The resulting terminated chains are resolved by electrophoresis
- Fragments from each of the four tubes are placed in four separate gel lanes
- Sequencing gels are read from bottom to top (5′ to 3′)
