APPLICATION OF PCR

&

VARIENTS OF PCR

ISOLATION OF A GENE BY PCR

If the Primers anneals anneal both the sides of the gene of interest

No selection required

THEN WHY CLONING

Sequence information is required

Length of DNA sequence

IF SEQ INFORMATION IS NOT KNOWN

If we have information on heterologous gene / equivalent gene

Its important to isolate gene whose seq is known

Fish out gene - based on flanking sequence information

DNA polymorphism

Allelic forms of genes can be isolated

Diagnostics – both in plant and animal including human - Early diagnosis

Forensic

We covered

Hot start PCR

Nested PCR

Touch down PCR

Overlap extension

Allele Specific PCR/ PASA

Asymmetric PCR

Multiplexing

….. and

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

• Steps in the RT-PCR reaction:– RNA isolation– Reverse transcription– PCR amplification– Analysis of PCR product

Reverse Transcription of RNA (RT)

• Primer Options for RT reaction– Oligo (dT)– Random Hexamers– Sequence-specific Primers

• Enzyme Options for RT Reaction– Retroviral RNA-directed DNA polymerase– AMV Reverse Transcriptase (avian myeloblastosis virus)– MMLV Reverse Transcriptase (Moloney murine leukemia

virus)

Real Time PCR (RT-PCR)

qPCR

A DNA-binding dye binds to all double-stranded (ds) DNA in PCR, causing fluorescence of the dye.

An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentrations to be quantified.

However, dsDNA dyes such as SYBR Green will bind to all dsDNA PCR products, including nonspecific PCR products (such as Primer dimer). 

Fluorescent reporter probe method

A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction

LATE-PCR

Smiths Detection has an exclusive license for LATE-PCR from Brandeis University for all markets, worldwide

This enables one to detect low numbers of target organisms

Assembly PCR / Polymerase cycling assembly ( PCA)

A method for the assembly of large DNA oligonucleotides  from shorter fragments.

The process uses the same technology as PCR, but takes advantage of DNA hybridization and annealing as well as DNA polymerase to amplify a complete sequence of DNA in a precise order based on the single stranded oligonucleotides used in the process.

It thus allows for the production of synthetic genes and even entire synthetic genomes.

The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product

The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product

Inverse PCRCommonly used to identify the flanking sequences around genomic inserts. It involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.

Ligation-mediated PCR: uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA foot printing

LM-PCR is a method of sequencing directly from genomic DNA if you have a primer for the region of interest.

The DNA is chemically cleaved as if for Maxam Gilbert chemistry, producing a whole bunch of fragments that end in G, A, T or C.

Denature the DNA and let the primer anneal to the fragments that are related the region you are interested in.

By primer extension off that primer, you make only those fragments double stranded and blunt ended; these molecules are able to be ligated to a linker (the linker is staggered at one end, and blunt on the other, so the ligation is directional

Now you have a bunch of fragments that have two known ends

(your primer, and the linker) and you can use PCR to amplify the various fragments, run them on a gel and determine the sequence.

The reason you might want to go to all this trouble would be to examine conditions of DNA that are lost upon cloning (e.g. methylation), or to examine footprints of DNA binding proteins in vivo.

Methylation-specific PCR (MSP): In higher order eukaryotes, DNA is methylated only at cytosines located 5' to guanosine in the CpG dinucleotide.

This modification has important regulatory effects on gene expression, especially when involving CpG-rich areas known as CpG islands

MSP used to detect methylation of CpG islands in genomic DNA.

DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine.

Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences.

At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA.

MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.

Solid Phase PCR:

Polony Amplification (where PCR colonies are derived in a gel matrix, for example),

Bridge PCR (primers are covalently linked to a solid-support surface)

Conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers)

Enhanced Solid Phase PCR (where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thermal 'step' to favour solid support priming)

BRIDGE PCR

Thermal asymmetric interlaced (TAIL PCR): Was developed by Liu and Whittier in 1995

For isolation of an unknown sequence flanking a known sequence.

Within the known sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures; a degenerate primer is used to amplify in the other direction from the unknown sequence

Specific primer (SP)

„Nested sequence specific primer complementary to vector sequence„ High melting temperature, Tm=58-63 deg. C

Arbitrary degenerate (AD) primer

Relatively shorter

Lower melting temperature, Tm =47-48 deg. C

Universal Fast Walking: for genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific primer and one general primer — which can lead to artefactual 'noise‘)

RAPID Amplification of cDNA Ends (RACE)

In silico PCR:

 (Digital PCR, Virtual PCR, Electronic PCR, e-PCR) refers to computational tools used to calculate theoretical polymerase chain reaction results using a given set of primers(probes) to amplify DNA sequences from a sequenced genome or transcriptome

DD-PCRDD-PCR

To detect the differential expression of the gene

Usually subtractive-hybridization and differential cDNA cloning require large amounts of mRNA derived from cell or tissue samples, and are very laborious and time consuming, often taking many months from start to finish.

AlternativelyDifferential Display – PCR: requires only small amounts of cell or tissue samples, and can be completed within

weeks.

The DNA from these gels slices is recovered by boiling, and reamplified by PCR using the original primers. The amplified DNAs are characterized by sub-cloning, sequencing and data-base searches

To verify the differential expression status of each of these sub-clones, Northern-Blot assays are performed using RNA isolated from the original sample sources

Differentially expressed cDNAs are easily visualized following autoradiography

COLONY PCRCOLONY PCR

Colony PCR can be used to identify colonies where your favorite gene (yfg) has been replaced with a marker gene by homologous recombination, and to distinguish homologous recombination events from non-homologous. It can also be used to identify colonies from a tetrad that carry a particular gene replacement

Dial Out PCR:

A highly parallel method for retrieving accurate DNA molecules for gene synthesis. A complex library of DNA molecules is modified with unique flanking tags before massively parallel sequencing. Tag-directed primers then enable the retrieval of molecules with desired sequences by PCR

DNA ligase

• Repairs gaps in the sugar-phosphate backbone of DNA

• Creates phosphodiester bonds

• Does not do anything with the bases

LIGASE CHAIN REACTIONLIGASE CHAIN REACTION

Two oligonucleotide probe hybridise side by side

A thermostable DNA ligase seal the nick

Each ligated product as well as original target serve as a template

A modification of this technique – gapped LCR (G-LCR)

Two oligonucleotide probe hybridise side by side

A thermostable DNA ligase seal the nick

Each ligated product as well as original target serve as a template

A modification of this technique – gapped LCR (G-LCR)

Kits are available for the detection of C.trachomatis, N. gonorrhoeae etc.

LCRLCR

The ligase chain reaction (LCR) is a method of DNA amplification. While the better-known PCR carries out the amplification by polymerizing nucleotides, LCR instead amplifies the nucleic acid used as the probe. For each of the two DNA strands, two partial probes are ligated to form the actual one; thus, LCR uses two enzymes: a DNA polymerase and a DNA ligase. Each cycle results in a doubling of the target nucleic acid molecule. A key advantage of LCR is greater specificity as compared to PCR

Helicase-dependent amplification

Similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles

DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation

TRANSCRIPTION MEDIATED AMPLIFICATION

OR

NUCLEIC ACID SEQUENCE BASED AMPLIFICATION

TRANSCRIPTION MEDIATED AMPLIFICATION

OR

NUCLEIC ACID SEQUENCE BASED AMPLIFICATION

Isothermal amplification techniques

RNA to DNA and DNA as a template for multiple copies of RNA (Like the life cycle of retrovirus)

Isothermal amplification techniques

RNA to DNA and DNA as a template for multiple copies of RNA (Like the life cycle of retrovirus)

TMA/ NASBATMA/ NASBA

Detection of HIV, HCV, vericella zoster virus, cytomegalovirus, rhinivirus, measles, papillomavirus, M. tuberculosis, M. pneumoniae etc.

STRAND DISPLACEMENT AMPLIFICATIONSTRAND DISPLACEMENT AMPLIFICATION

Isothermal amplification technique

To detect trace amount od DNA or RNA of a particular sequence

Detection of tuberculosis, trachomatis, gonorrhoea

Isothermal amplification technique

To detect trace amount od DNA or RNA of a particular sequence

Detection of tuberculosis, trachomatis, gonorrhoea

STRAND DISPLACEMENT AMPLIFICATIONSTRAND DISPLACEMENT AMPLIFICATION

5΄-GTPy↓PuAC-3΄

3΄-CAPu↑PyTG-5΄

SDASDA

Loop-mediated Isothermal Amplification (LAMP) PCR

Dieffenbach, C.W and Dvksler, G.S. (1995) PCR primer: a laboratory manual. CSHL press, Cold Spring Harbor, USA.

Erlich, H. A. 1989. PCR technology: Principles and applications for DNA amplification. Stockton Press, New York.

Innis, M. A., D. H. Gelfand, J. J. Sninsky, and T. J. White. 1990. PCR Protocols: A guide to methods and applications. Academic Press, New York.

Ellingboe, J., and U. B. Gyllensten. 1992. The PCR technique: DNA sequencing. Eaton Publishing Co., Natick, Mass.

Bej, A. K., M. H. Mahbubani, and R. M. Atlas. 1991. Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications. Critical Reviews in Biochemistry and Molecular Biology 26:301-334.

Journal:

"PCR Methods and Applications" published quarterly by Cold Spring Harbor Press

REFERENCEREFERENCE