An Overview of the PCR

“The polymerase chain reaction (or PCR) is a technique for the in vitro

amplification of a desired sequence of DNA”

• PCR allows the generation of a large quantity of DNA product from only a few starting copies. It has been shown that PCR can be used to generate a detectable quantity of DNA from only one starting target (or template) molecule.

PCR was developed in the mid-1980's, but has already found multiple applications, such as:

1. Rapid amplification of intact genes or gene fragments

2. Generation of large amounts of DNA for sequencing

3. Analysis of mutations for medical applications

4. Amplification of chromosomal regions adjacent to genes of known sequence

And many more·

Development of PCR won the Nobel prize for Kary Mullis and co-workers.

PCR….

1. PCR allows the specific synthesis of a predetermined DNA region via the use of two small, specifically designed fragments of DNA (primers or oligonucleotides),

2. two termini nucleic acid molecule amplified. 3. PCR amplification reactions in general are highly

specific, specificity being determined by the correct hybridisation of primer specific sequences to complementary sequences present on the target DNA molecule to be amplified

steps in PCR• There are three major steps in a PCR, which

are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time.

1. Denaturation at 94°C : all enzymatic reactions stop (for example : the extension from a previous cycle).

2. Annealing at 54°C :Brownian motion.

• Ionic bonds are constantly formed and broken

• The more stable bonds …..on that little piece of double stranded DNA …. the polymerase can attach….starts copying the template.

3. extension at 72°C :ideal working temperature for the polymerase.

• The primers……already have a stronger ionic attraction to the template than the forces breaking these attractions.

• Primers that are on positions with no exact match, get loose again

• The bases (complementary to the template) are coupled to the primer on the 3' side

PCR theory

“The PCR reaction is a DNA synthesis reaction that depends on the extension of primers annealed to opposite strands of a dsDNA template that has been denatured (melted apart) at temperatures near boiling. By repeating the melting, annealing and extension steps, several copies of the original template DNA can be generated”

Brief Overview of PCR Requirements

PCR Reaction Mix

1) Target DNA 2) specifically primers 3) Deoxyribonucleotide triphosphates4) magnesium ions5) Thermostable DNA polymerase (Thermus

thermophilus, T. flavus, T. litoralis, T. brokianus)6) water. 7) RT (Avian Myeloblastoma Virus (AMV) and Moloney

Murine Leukemia Virus (MMLV)

All prepared in a total typical PCR reaction volume of 20-50 μl

PCR Con…..1. composition of the PCR reaction mix may vary

dependant on the DNA polymerase used2. Low concentrations of detergents such as Triton X-

100, Tween-20, betain or dimethylsulphoxide (DMSO) may also be included in the PCR mix to help increase the specificity of primer binding.

3. DMSO may assist in overcoming problems caused by secondary structure or possibly even inhibitory compounds.

4. The composition of the PCR mix should ideally be optimized

5. All PCR reaction ingredients should be stored in a freezer in a dedicated “clean” room

6. multiple PCRs in a single “batch”

Miscellaneous Considerations

1. Several additional factors ….Success or failure of PCR

2. Nature and use of the PCR reaction tube

3. Changes in the quality of ingredients affect the sensitivity and specificity of the PCR reaction

4. Controls are particularly important

DNA Extraction /RNA Extraction

The Different Types and Varietiesof Nucleic Acid Target Molecules

• must be readily accessible to primers and DNA polymerase and free from inhibitory concentrations of contaminating proteins, lipids, carbohydrates and salts

• Blood samples• RNA• DNA

• Serum samples

A Brief Description of DNA and RNA Targets

A.BacteriaB.Viruses

1.Class I: Double stranded DNA viruses. human papilloma viruses (cervical cancer). Replicate in the host cell cytoplasm.

2.Class II: Single stranded DNA viruses One example is Parvovirus B19

3.Class III: Double stranded RNA viruses example is rotaviruses

4.Class IV: single stranded RNA viruses example is Poliomyelitis virus

5.Class V: single stranded RNA viruses, Influenza viruses

6.Class VI: Retroviruses, HIV

Contaminations….• Blood

• Inhibitors• kits

• RNase

1.gloves should be worn2.use of disposable RNase free plastic…….3.work surfaces are also …RNase free4.inactivated by….. DEPC (diethyl pyrocarbonate) or

commercially available …… RNase AWAY, RNase ERASE, RNA Clean

5.DEPC in water being used to remove RNase6.DEPC also hydrolyses the imidazole ring of purines and

destroys nucleic acids.7.180°C to inactivate the DEPC8.DEPC….toxic carcinogen that……….safety precautions

• DNase

Primers

Primers

• Hybridisation• provide the 3′ hydroxyl ends• position of the primers along DNA strands

defines length of the PCR amplification product generated Primers

• PCR amplification products (100–200 nucleotides), long PCR amplification products (3,000–5,000 bp)

• primer annealing sites on the target DNA must be known

• 18–22 nucleotides

Primers…..

1. hybridise to either the parallel or anti-parallel strand

2. need to be precisely complementary to their target sequences, some sequence data from the terminal ends of the DNA is required for primer design (Fig. 1.1).

3. Once hybridised …. 3¢-hydroxyl terminus required by DNA polymerases

4. effectively acting as Okazaki fragments

PCR Primer Design and Quality Requirements

1. base composition2. length of primer3. GC-content4. annealing temperatures,5. no internal complementary regions6. Complementary regions…..primer/primer

hybridisation…… “primer dimer”7. secondary structure….. primer dimers8. primer-dimer formation…..multiplex PCR protocols,9. Y=G, z=C, x=T, and w=A

main rules for effectiveprimer design

• 18 and 30 nucleotides (bp) in length.• no internal complementary regions (e.g.

multiple guanosine and cytosine repeats)• GC content 50%• genetically stable….. Conserved sequences• inosine nucleotide• annealing temperatures…65°C• annealing temperatures ….. ±5°C

Primer Hybridisation Depend (Annealing)

• annealing time….dependent on the ramp rate• heating capacity…..PCR thermocycler used• volume of the PCR mix• the concentrations of the primer and target

template• composition of the sample buffer• primer length• GC content• reaction tubes• Tm = 2 (T+A) + 4 (G+C)

Primer Synthesis

• Commercial companies

• nucleic acid synthesising machines (e.g. the ABI 3400 DNA synthesizer, USA).

Effect of Mismatches Between PCRPrimer and Target

1. Mutations in the target sequence or incorrectly designed PCR primers

2. resulting in incomplete base pairing between the two nucleic acid molecules….. lowers the annealing temperature (Tm)……reducing the yield of specific PCR amplification products

3. base pair mismatches 3¢-end of the primer …… larger impact than those near the 5¢-end of the primer

Primer Concentration

• 0.1–1 μM concentration ….. (in 50 μl)

• but excess of primer…….non-specific PCR products so

• Increasing annealing temperature• lowering the initial primer concentration • primer concentration versus magnesium ion

concentration

Deoxynucleotide Triphosphates(DNTPs)

DNTPs….1. building blocks of nucleic acid molecules2. necessary components of PCR mixes3. The four individual deoxynucleotides…in PCR

1. Deoxyadenosine triphosphate, dATP2. Deoxythymidine triphosphate, dTTP 3. Deoxycytosine triphosphate, dCTP 4. Deoxyguanosin triphosphate, dGTP

4. purchased either individually or as an mix5. stable when stored at −20°C6. Acidic in nature…so...working and stock solutions …. to

be neutralized with alkaline compounds7. 10 mM stock solutions8. dNTPs may be lost due to non-specific heat

inactivation…… during a 40 cycle PCR program….. loss of dNTPs may be in 50% of the original amount added to the PCR mix

DNTPs….

• dNTPs…pH 7.2...negative charge….ability to bind both mono- and divalent cations…..mg ions

• Mg ions …. bound by dNTPs

Factors Affecting the Choice of dNTP Concentration

• use a concentration of dNTPs …. only a fraction at the end of the PCR cycling

• if the dNTP limiting...rate of dNTP incorporation will be reduced

• 10μl……..0.2 μl

Modified dNTPsTypical PCR protocol….all four dNTPs1. specially modified dNTPs may also be added to

the PCR2. helix destabilizing nucleotide>>7-deaza- 2′-

deoxyguanosine3. Inosine is a naturally occurring nucleotide

structural build in block tRNA4. capable of base pairing with any of the four

dNTPs5. helps to overcome amplification problems6. solve problems related to sequence variation

within the target DNA.7. useful in amplifying CpG islands (GC rich

sequence found in the promoter sequences of many higher eukaryotes)

Disadvantage of the use ofModified dNTPs• 7-deaza-2′-deoxyguanosine is very difficult

to stain using ethidium bromide.

• So dGTP is also added to the PCR mix at a concentration 25%

• their presence modifies restriction digest recognition sites…. these sites may no longer be recognized by restriction enzymes.

M-nucleoside……

• Alternatively, another universal nucleoside (1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrole) has been described in the literature

• This nucleoside is known as “M-nucleoside”• maintains the ability …. four normal dNTPs• its incorporation in PCR primers affects the

Tm of primer annealing much less markedly than inosine

The PCR Buffer

PCR Buffer

• (10X concentrated) PCR reaction buffer

• DNA polymerase activity…pH conditions should be maintained

• ammonium sulphate help to remove any inhibitory products, such as pyrophosphate, ….. accumulate during PCR amplification.

Monovalent Ions

Monovalent Ions……

• Sodium (Na+), potassium (K+) and ammonium (NH+4) ions stimulate the activity of polymerases

• Potassium ions have an optimum stimulatory effect on PCR DNA polymerases

• NH+ 4 ions compete for the hydrogen bonds

Divalent

Magnesium Ions……• important ingredient of PCR reaction• co-factor for thermostable DNA polymerase activity,

stimulating the enzymes• optimization is frequently performed in combination

with an experiment to determine the optimum dNTP concentration

• magnesium ion concentration range of between 0.5 and 5 Mm

• elevated magnesium ion concentrations inhibit PCR……double stranded DNA …actually stabilized ….

• increasing the risk of nonspecific product amplification

Taq and Other ThermostableDNA Polymerases

DNA Polymerases……

DNA polymerase….the success of PCR• Klenow fragment of DNA-dependent DNA polymerase

I…. disadvantage • 1 lacks a 5¢-3¢ exonuclease activity....• optimum reaction temperature at 37°C…• impossible to generate PCR….longer than 400 bp

Taq polymerase

“in 1976 Chien described a 94kD thermostable DNA-dependant DNA polymerase derived from a eubacterium called Thermus aquaticus”

3¢-5¢ proofreading domain…. missing

The Advantages and Disadvantages of Taq over

Klenow Fragment DNA Polymerase

Advantages…1. Heat stable … half-life of 130 minutes at a temperature of 92.5°C.2. Reaction vial not… opened in order to add fresh enzyme …

reducing contamination3. optimum reactivity …. 70°C-80°C…helps to prevent any secondary

or tertiary structure4. High optimum temperature …prevent mispriming of the PCR

primer5. Has increased processivity at elevated incubation temperature.6. Capacity to synthesize relatively long PCR products…4,000 bp

(8,000–10,000 DNA quality)7. Generates significantly higher yields of amplification products (ten

times higher) 60 nucleotides per second at 72°C, but– only 1.5 nucleotides per second at 37°C

8. Enzyme is not inhibited by chemical contaminants remaining after nucleic acid extraction, e.g. chloroform

9. Taq enzyme frequently adds an overhanging nucleotide

Prevent mispriming of the PCR primer

Optimum reactivity …. 70°C-80°C

Disadvantageous of Taq polymerase 1. Susceptible to proteolytic degradation2. Taq polymerase inhibited … wide variety of compounds which include:-

1. >2% phenol2. 1% ethanol,3. DMSO, 4. EDTA5. Guanidinium HCl6. urea7. agar and agarose.8. 1% isopropanol9. >5 mM sodium acetate (NaAc)10. bromide (CTAB),11. 0.005% sodium dodecyl sulphate (SDS), 12. >20% formamide,13. RNase inhibitor 14. blood anticoagulant (polyanetholsulphate)15. Plant polysaccharides such as glycan, acid mucopolysaccharides,

polyphenols, rice starch, pectin, dextransulphate and b-glucans

Analysis of PCR Amplification Products

Gel Electrophoresis

(i) Simple agarose gel electrophoresis(ii) Simple polyacrylamide gel electrophoresis (PAGE)(iii) Restriction Fragment Length Polymorphism (RFLP)

analysis and oligomer restriction(iv) Single Stranded Conformation Polymorphism (SSCP)

analysis(v) Denaturing Gradient Gel Electrophoresis (DGGE)(vi) Image analysis of PCR amplification products in gel

electrophoresis systems(vii) Excising and cleaning PCR amplimers from

electrophoresis gels

Simple agarose gel electrophoresis

1. polysaccharide agarose (poly d-galactose 3,6-anhydro-l galactose

2. purified from marine algae

3. solid compound at room temperature…. powder

• Agarose matrix … molecular sieve• Electric current … opposite ends of the gel .. migration of –ve

DNA molecules to the +ve anode in the electrophoresis tank• Average agarose gel … 0.8 and 4% agarose• 1–2% .. separation of PCR amplimers … 100bp• Does not allow …separation of molecules less than

approximately 15 nucleotides in length.• Higher the agarose concentration more dense polymer

network

Type of electrophoresis buffer

Buffer used … affect the DNA separation

1. TAE (Tris-Acetate-EDTA) ..1. Resolution of fragments larger than 4 kb

2. TBE (Tris-Borate-EDTA) ..1. providing better resolution of 0.1–3 kb fragments

2. Reused a few times

Ladders…. Amplimer size

determination…. molecular weight markers … added to an empty well

PCR Failures… Smear of amplification products…. may be caused by….

1. Degraded PCR primers, Contamination from previous PCRs,

2. Excessive quantity of Taq

3. High magnesium ion concentration

4. Number of cycles in the PCR

5. Amount of template DNA

6. DNA added to the PCR mix may be impure or degraded………