Pathogens Pcr

Pathogens & PCR Technology

DNA REPLICATION „ „ „ „

B.K.Kolita Kamal Jinadasa, Jinadasa, Post Harvest Technology Division, NARA, ColomboColombo-15, Sri Lanka.

DNA REPLICATION „

The most important enzyme of the DNA replication is DNA Polymerase I (Pol I). Three activities are associated with DNA polymerase I; 5' to 3' elongation (polymerase activity) 3' to 5' exonuclease (proof(proof-reading activity) 5' to 3' exonuclease (repair activity)

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The replication of DNA in vitro is named polymerase chain reaction reaction (PCR). PCR is used to amplify specific regions of a DNA strand. strand. With this tecnique we can have a lot of copy of a determinate region. region.

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PCR is especific bacause we amplify specific regions of bacterias, animals, animals, plants, plants, viruses... viruses... Thes regions are named molecular markers. markers.

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For example, example, if we amplify a genetical marker of E.coli, E.coli, we are sure that only E.coli will be amplify and the others bascterias don’ don’t amplify. amplify.

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PCR was invented by Kary Mullis. Mullis. At the time he thought up PCR in 1983, Mullis was working in Emeryville, Emeryville, California for Cetus Corporation .

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The polymerase chain reaction was introduced to the scientific community at a conference in October 1985

Kary Mullis

When a cell divides the extra DNA comes from replication DNA replication only occurs at a specific step in the cell cycle DNA replication is semisemi-conservative, one strand serves as the template for the new strand One of the parent strands of DNA is 3' -> 5' and the other is 5' -> 3'. To solve this replication is in opposite directions. Heading towards the replication fork, the leading strand in synthesized in a continuous fashion, only requiring one primer. On the other hand, the lagging strand, heading away from the replication fork, is synthesized in a series of short fragments known as Okazaki fragments, consequently requiring many primers

DNA strands

PCR: POLYMERASE CHAIN REACTION .

Nobel Prize for chemistry in 1993

Thermocycler

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PCR, as currently practiced, practiced, requires several basic components: components:

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DNA template that contains the region of the DNA fragment to be amplified. amplified.

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Taq polymerase (or another DNA polymerase with a temperature optimum at around 70° 70°C), a DNA polymerase, polymerase, used to synthesize a DNA copy of the region to be amplified. amplified.

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Deoxynucleotide triphosphates, (dNTPs) dNTPs) from which the DNA polymerase

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Buffer solution, which provides a suitable chemical environment for optimum

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Divalent cation, cation, magnesium or manganese ions; ions; generally Mg2+ is used, used, but

builds the new DNA.

activity and stability of the DNA polymerase. polymerase.

Mn2+ can be utilized for PCRPCR-mediated DNA mutagenesis, mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis. synthesis.

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PCR STEPS „

PCR steps

The PCR usually consists in a series of 20 to 35 cycles. cycles. PCR is carryed out in three steps: steps: 1.

Denaturation at 94° 94°C : During the denaturation, denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for (for example: example: the extension from a previous cycle). cycle).

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The denaturation is followed by the annealing step. step. In this step the reaction temperature is lowered so that the primers can attach to the singlesingle-stranded DNA template. template. The temperature at this step depends on the Tm of the primers (see above), above), and is usually between 5050-64° 64°C for 2020-40 seconds. seconds.

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The annealing step is followed by an extension/ extension/elongation step during which the DNA polymerase copies the DNA template, template, starting at the primers annealed to both of its strands. strands. The temperature at this step depends on the DNA polymerase used. used. Taq polymerase has a temperature optimum of 70thus, in most cases, during the extension a 70-74° 74°C; thus, temperature of 72° 72°C is used

PCR

General PCR

One PCR cycle consists of the following steps „

Initialization. Initialization. The mixture is heated at 96° 96°C for 5 minutes to ensure that the DNA strands as well as the primers have melted. melted.

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Melting, Melting, where it is heated at 96° 96°C for 30 seconds. seconds. For each cycle, cycle, this is usually enough time for the DNA to denature. denature.

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Annealing by heating at 68° 68°C for 30 seconds. seconds. Stable bonds are only formed when the primer sequence exactly fits the template sequence, sequence, and on that short piece of doubledouble-stranded DNA (template (template and primer), the polymerase can attach and start copying the template. template. Once this extension has created a longer doubledouble-stranded DNA segment, segment, the Tm of this doubledouble-stranded region is now greater than the annealing or extension temperature.

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Elongation by heating 72° 72°C for 45 seconds: seconds: This is the ideal working temperature for the polymerase. polymerase. The combined hydrogen bonds between the extended primer and the DNA template are now strong enough to withstand forces breaking these attractions at the higher temperature. temperature. Primers that are on positions with no exact match, melt away from the template (because of the higher temperature) temperature) and are not extended.

Exponencial amplification

TYPES OF PCR „ 1. 2. 3. 4. 5. 6. 7. 8.

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Because both strands are copied during PCR, there is an exponential increase of the number of copies of the gene. Supposing that there is only one copy of the wanted gene before the cycling starts, starts, after one cycle, cycle, there will be 2 copies, after two cycles, cycles, there will be 4 copies, three cycles will result in 8 copies and so on. on.

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There are a lot of types of PCR : Nested PCR Inverse PCR RTRT-PCR (Reverse Transcription PCR) Quantitative realreal-time PCR MultiplexMultiplex-PCR Asymmetric PCR "Hot"Hot-start" PCR PCRPCR-RFLP PCRPCR-ELISA

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APLICATIONS OF PCR „

PCR is commonly used in medical and biological research labs for a variety of tasks, tasks, such as the detection of hereditary diseases, diseases, the identification of species, species, diagnosis of infectious diseases, diseases, cloning of genes, genes, paternity testing, testing, DNA computing and detection of pathogens in food. food.

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The detection of foodborne pathogens is very important bacause this pathogens can cause very important illnes. illnes.

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Some of foodborne pathogens that we can find in food are: Escherichia coli, coli, Salmonella spp, spp, Shigella spp,, Staphylococcus aureus,, Vibrio choleraea and Vibrio parahaemolyticus.

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