Lesson 14. PCR AND REAL TIME PCR

Module 3. Genetic engineering technology / recombinant DNA technology

Lesson 14
PCR AND REAL TIME PCR

14.1 Polymerase Chain Reaction (PCR)

PCR is one of the most powerful tools in Molecular Biology. It is defined as a technique used for in vitro amplification of specific target DNA sequence using a set of specific and unique primers for primer extension of complementary strands by Taq Polymerase. PCR acts like a ‘copying or xeroxing machine’ creating a large number of duplicated copies of DNA molecules from a minute amount of starting material. It is a rapid, inexpensive, extraordinarily powerful and most commonly used versatile technique in all day to day lab experiments. Kary Mullis from Cetus Corporation was awarded Nobel Prize in chemistry in 1993 for his invention of PCR in 1983.

PCR reaction consists of the following components as depicted in Fig. 14.1.

These are briefly described as follows:

14.1.1 Template / Target DNA

Template DNA is the resource material for the amplification of the target gene. It is extracted by following different DNA extraction protocols. It could be either genomic DNA, plasmid DNA or cDNA prepared from RNA from any source like microbes, viruses, animals and plants. The beauty of this technique is such that because of its high specificity, it can pick up the target amongst the large background of the non-specific DNA also and hence template DNA need not be absolutely pure for PCR amplification.

14.1.2 Deoxynucleotide triphosphates (dNTPs)

The deoxynucleotide triphosphates (dNTPs) are the nucleotide building blocks (adenine, guanine, cytosine and thymine) which need to be present for incorporation during amplification of the target gene.

14.1.3 Primers

A pair of primers (Forward and Reverse) is required for amplification of the target DNA from both the strands i.e. 5’---3’ and 3’----5’. In brief, primer is a short single-stranded oligonucleotide sequence of DNA that is required to initiate the synthesis of new strand of DNA in a polymerase chain reaction ( Fig._14.2.swf & Fig. 14.3). Generally, these are about 20 to 22 bp in length for general PCR reaction but may vary according to the designed experiment (upto even 50 bp). The primers are identical to the 5’ ends of sense and antisense strands of DNA. These are designed to flank the terminal regions of gene to be amplified as shown below also:

14a

14 d

Melting and annealing temperatures are the two most important parameters which need to be looked into while designing a primer for their optimal usage.


Melting and annealing temperatures of primers can be determined by the following formulae :

Tm (oC) - 4 (G + C) + 2 (A+T)

Annealing temperature i.e. Tanneal = Tm - 4oC

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14.1.4 Taq DNA polymerase

Most of the enzymes/proteins get inactivated at higher temperature but Taq DNA polymerase withstands high temperature of denaturation. Taq DNA polymerase was isolated from a thermophilic organism Thermus aquaticus, which normally lives in hot springs at temperatures close to 100°C. Hence, this enzyme can remain stable at high temperatures as close as 100°C i.e. it is stable under the extreme temperature conditions of PCR and hence does not need to be supplied afresh in the PCR reaction mix. Taq DNA polymerase catalyses DNA polymerization i.e incorporates / adds nucleotides during the synthesis of new strands of DNA during extension at 72°C. However, Taq DNA polymerase requires the use of a buffer containing MgCl2 for its optimal functionality. Similar enzymes have been isolated form other thermophilic bacteria which even possess proof reading activity (Pfu polymerease) and are used in cloning of genes to avoid any errors. Many companies are now selling recombinant Taq polymerase which is very cheap and considerably economical .

14.1.5 Thermal cycler

The Thermal cycler also known as PCR machine or DNA amplifier is an equipment which is used for amplification of a gene using polymerase chain reaction. These thermal cyclers are available form a number of companies viz. Applied Biosystems, Bio-Rad, Eppendorf, Perkin Elmer, MJ Research Inc. etc. The machine consists of a thermal block whose temperature is raised or lowered according to the programmed steps during cycling parameters. These machines also come with heated lid to prevent condensation of water from reaction mix. Some of the machines have silver blocks which can achieve fast temperature changes and uniform temperature through out the block.

14.1.6 Cycling parameters

PCR cycling parameters involve several cycles (25 – 40) of denaturation, annealing and extension in order to amplify a desired gene.

14.1.6.1 Denaturation

Template DNA is heated at high temperatures (94°C) at which the duplex DNA gets depolymerized to single strands. These strands then become accessible to primers annealing for synthesis of new strands during amplification (Fig. 14.3A).

14.1.6.2 Annealing

The reaction mixture is cooled down to the specific temperature (~ 55°C) at which the primers anneal to the complementary regions in the template DNA.

14.1.6.3 Extension

Taq DNA polymerase then starts adding nucleotides to the respective primer using information from the DNA template strand at 72°C.

These steps are followed again and again to generate multiple copies of the DNA template i.e amplified PCR product or amplicons.

Initially, an initial denaturation step at 94°C for 2-5 min. is given followed by 30-40 cycles of denaturation at 95°C/30sec to 1 min.; annealing at 45-65°C/30 sec to 1 min. and extension at 72°C for 30 sec to 2 min. depending on the length of fragment to be completed and final extension step at 72°C for 5-10 min. The amount of amplified product at the end of PCR cycles is 2n# of cycles e.g. if we start with a single molecule of DNA, after 25 cycles, the amount will be 225 i.e. 3.4x107 molecules of DNA.

14.2 Analysis of PCR products

The specificity of PCR products is ascertained by any of the following protocols:

14.2.1 Agarose gel electrophoresis

The PCR products are electrophoresed along with molecular size markers i.e. 100 bp, 250 bp, 500 or 1 kb ladder or any other desired size marker to check the specificity of the desired amplicon. The presence of a PCR product specific / unique for a particular target gene indicates the presence or detection of that gene in an organism e.g. specific sizes of 147 for gapB,123 for ldhD etc. are detected as shown in the Fig. 14.4.

14.5

Fig. 14.4. Agarose gel electrophoresis of PCR products


14.2.2 Restriction analysis

The PCR products are digested with the restriction enzymes to yield specific fragments of desired length for confirmation of the amplified gene.

14.2.3 Southern hybridization

It involves the application of a radio-labeled or non-radio-labeled DNA probes (nucleotide sequence of upto 50 bases for the target gene) to check for the specificity of the PCR product.

14.3 Applications of PCR

PCR finds applications in almost all the branches of science and areas and in a variety of other fields such as
  • Diagnostics
Detection of food borne pathogens from dairy foods, water and other food products as well as clinical samples
  • Forensic Labs – paternity testing
  • In vitro mutagenesis (Site directed mutagenesis)
  • Molecular Evolution
  • Sequencing
  • Cloning of genes
  • Monitor cancer therapy
  • To detect mutations ( genetic disorders)
  • Metagenomics
14.4 Reverse Transcriptase –PCR (RT-PCR)

RT-PCR, the Reverse Transcriptase PCR involves two steps viz. Reverse Transcriptase step and PCR amplification. RNA is extracted and is reverse transcribed into cDNA (complementary DNA) using the enzyme reverse transcriptase and polyT oligo or random or sequence specific primers. The cDNA is then used as a template for PCR amplification using specific sets of primers for amplification of the desired gene of interest. RT-PCR is a sensitive technique for detection of low copy number of mRNA molecules (the functional part of DNA). Detection of mRNA is used for gene expression studies and cloning of eukaryotic genes which possess introns.

14.5 Real Time PCR or qRT-PCR (quantitative RT-PCR or RT-qPCR) or qPCR or RTi-PCR

Real Time PCR was first reported by Higuchi et al (1992). It is also known as kinetic PCR or qPCR. Real Time PCR is based on detection of fluorescent signal. The Real Time PCR machine incorporates an optical module to detect fluorescence. Furthermore, compared to PCR, it is quantitative i.e. copy number of the gene can be determined. RT-PCR monitors the amplicon in real time by making use of reporter and quencher dyes. The most common reporter dyes are FAM, TAMRA, TET, HEX, Cy5, Cy3 etc. and Quencher dyes are TAMRA, DABCYL, BHQ-1 and BHQ-2 etc. The Real Time PCR machines available in market are from Applied Biosystems (ABI 7500, 7700, 9700 etc.); Bio-Rad (iCycler IQ); Stratagene (Mx 4000); Smartcycler II (Cepheid); Roche (Lightcycler 480) etc. All the machines incorporates a thermal cycler, a computer, optics for fluorescence excitation, data acquisition and analysis software. The most common Real Time PCR used all over the world are shown in the Fig. 14.5.

14.5a

Real Time PCR works on two types of chemistries viz. non-specific and specific which are discussed in the following section:

14.5b

Fig. 14.5 Range of Real time – PCR Instruments (Click for Animation)

14.5.1 Non-specific chemistry

Non-specific chemistry involves using intercalating dyes which bind to all double stranded DNA produced during PCR reaction.

14.5.1.1 Intercalating dyes

Intercalating dyes are the ones which bind to any double stranded DNA generated during any PCR or Real Time PCR reactions. These dyes fluoresce once bound to the DNA. The most common dye is Syber Green (SYBR Green) which gets excited at 497 nm and emits fluorescence at 520 nm. The specificity of products can be determined by melt curve / dissociation curve analysis. The reaction mix is slowly heated from 40 to 95°C while continuously monitoring the fluorescence. The point at which double stranded DNA melts is recorded as a drop in fluorescence as the SYBR Green dissociates from ds DNA molecules. Every target gene sequence has its own specific melting point which can help in distinguishing different targets in one reaction. Primer dimers etc. can also be distinguished. Fig. 14.6 gives an outline of the intercalating chemistry.

14.5.2 Specific detection chemistries

There are a number of chemistries used in Real Time PCR which involve use of probes labeled with fluorescent / reporter and quencher dyes. Specific chemistries include Molecular Beacons, Scorpion probes, TaqMan probes, FRET probes, Amplifuor, Dual labeled probes etc. which discriminate between specific and non-specific DNA sequences. The commonly used chemistries are shown in Fig. 14.7.

Two of the Real Time PCR chemistries will be discussed here.

14.5.2.1 TaqMan / Hydrolysis probes

TaqMan probes also called hydrolysis probes or dual labeled probes are the most widely used probes. These were developed by Roche and ABI and consist of single stranded probe sequence that is complementary to one of the strands of amplicons. A fluorophore is attached at one end and quencher at the other end as shown in the following Fig. 14.8. The probe binds to amplicon during each annealing step of the PCR reaction. When Taq polymerase extends the target gene from the primer bound to the amplicon, it displaces the 5’ end of the probe which is then degraded by 5’ to 3’ exonuclease activity of Taq polymerase. This process separates the fluorophore and quencher and leads to irreversible increase in fluorescence which is being read in every extension step.

14.5.2.2 Molecular beacons

Molecular beacons MB) were invented by Tyagi and Krammer in 1996 and differ from the TaqMan probes. MBs consist of single stranded DNA with hairpin loop structure (Fig. 14.9). The loop which is single stranded bears complimentarity to the amplicon. The stem is approximately six to eight bases long and mainly consists of G and C which can hold the probe in hairpin structure. The stem bears fluorophore at one end and quencher at another. The probe opens up at high temperature and binds to specific sequence in the target DNA. Binding of probe to amplicon distrupts the stem loop structure leading to the separation of fluorophore and quencher resulting in fluorescence which can be read during annealing step.

Books

Molecular Biotechnology - Second Edition, S. B. Primrose, Blackwell Science Inc., ASIN: 0632030534

Introduction to Biotechnology, Brown, C.M., Campbell, I and Priest, F.G. Panima Publishing Corporation, 2005. ISBN : 81-86535-42-X

DNA and Biotechnology, Fitzgerald-Hayes, M. And Reichsman, F. 2nd Amsterdam : Elsevier, 2010. ISBN : 0-12-048930-5

Molecular Biotechnology : Principles and Applications of Recombinant DNA, Glick, B.R., Pasternack, Jack, J and Patten, Cheryl, L (Eds)., 4th Washington., ASM Press, 2010. ISBN : 1-55581-498-4

Molecular Biology and Biotechnology : a guide for students, Krauzer, H. And Massey, A.(Eds) 3rd Washington DC : ASM Press, 2008, ISBN : 978-155581-4724

Recombinant DNA and Biotechnology : a guide for teachers, Kreuzer, H and Massey, A. 2nd Washington : ASM Press, 2001, ISBN : 155581-175-2

Molecular Biotechnology, Primrose, S.B. 2nd New Delhi : Panima, 2001. ISBN : 81-86535-21-7

Molecular Biology and Biotechnology, Smith, C.A. and Wood, E.J. London : Chapman and Hall, 1991.ISBN : 0-412-40750-7

Introduction to Biotechnology, Thieman, W.J and Pallidino, M.A. 2nd New York : Pearson, 2009, ISBN : 978-0-321-58903-3

Molecular Biology and Biotechnology, Walker, J.M and Rapley, R. 4th – New Delhi : Panima Publishing Corporation, 2003, ISBN : 81-86535-40-3

Gene Biotechnology, Wu William, Welsh, M.J., Kaufman, P.B and Zhang, H.H. 2nd Boca Raton : CRC press, 2004. ISBN : 0-8493-1288-4

From Genes to Genomes: Concepts and Applications of DNA Technology, 3rd Edition, Jeremy W. Dale, Malcolm von Schantz, Nicholas Plant (Eds), Wiley-Blackwell, 2011, ISBN: 978-0-470-68386-6

Microbial Genetics, 2nd Edition, Stanly R Maloy, John Cronan, David Freifelder, Narosa, ISBN: 8173196974

Molecular Biology of the Gene, Sixth Edition, James D. Watson (Editor) Cold Spring Harbour Press and Benjamin Cummings, ISBN 978-080539592-1

Internet Resources

http://www.blackwellpublishing.com/trun/pdfs/Chapter12.pdf

http://en.wikipedia.org/wiki/Polymerase_chain_reaction

https://facultystaff.richmond.edu/~lrunyenj/bio554/lectnotes/chapter14.pdf

http://en.wikipedia.org/wiki/Real-time_polymerase_chain_reaction

http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Pierce/realtimepcr.htm
http://www.bio.davidson.edu/courses/genomics/method/realtimepcr.html





Last modified: Saturday, 25 August 2012, 6:17 AM