Procedures for PCR

The PCR usually consists of a series of 20 to 35 cycles. Most commonly, PCR is carried out in three steps (Fig. 2), often preceded by one temperature hold at the start and followed by one hold at the end.

1) Prior to the first cycle, during an initialization step, the PCR reaction is often heated to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used), and this temperature is then held for 1-9 minutes.

-This first hold is employed to ensure that most of the DNA template and primers are denatured, i.e., that the DNA is melted by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding two single strands of DNA.
-Also, some PCR polymerases require this step for activation (see hot-start PCR).[5] Following this hold, cycling begins, with one step at 94-98°C for 20-30 seconds (denaturation step).

2) The denaturation is followed by the annealing step.

-In this step the reaction temperature is lowered so that the primers can anneal to the single-stranded DNA template. Brownian motion causes the primers to move around, and DNA-DNA hydrogen bonds are constantly formed and broken between primer and template.
-Stable bonds are only formed when the primer sequence very closely matches the template sequence, and to this short section of double-stranded DNA the polymerase attaches and begins DNA synthesis. The temperature at this step depends on the melting temperature of the primers, and is usually between 50-64°C for 20-40 seconds.

3) The annealing step is followed by an extension/elongation step during which the DNA polymerase synthesizes new DNA strands complementary to the DNA template strands.

-The temperature at this step depends on the DNA polymerase used. Taq polymerase has a temperature optimum of 70-74°C; thus, in most cases a temperature of 72°C is used. The hydrogen bonds between the extended primer and the DNA template are now strong enough to withstand forces breaking these attractions at the higher temperature.
- Primers that have annealed to DNA regions with mismatching bases dissociate from the template and are not extended. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified.
-As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases in one minute.
-A final elongation step of 5-15 minutes (depending on the length of the DNA template) after the last cycle may be used to ensure that any remaining single-stranded DNA is fully extended.
-A final hold of 4-15°C for an indefinite time may be employed for short-term storage of the reaction, e.g., if reactions are run overnight.


~To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as amplimer), agarose gel electrophoresis is commonly employed for size separation of the PCR products. The size(s) of PCR products is thereby determined by comparison with a DNA ladder, which contains DNA fragments of known size, ran on the gel alongside the PCR products


*Extras: `Uses of PCR`
=>Isolation of genomic material

Part of PCR's power lies in its ability to easily isolate particular regions of DNA sequence from whole genomic material. Many techniques need a pool of DNA molecules isolated from a particular DNA fragment, and the use of PCR has enabled these techniques more widespread in usage. Because PCR also amplifies the isolated region, the techniques are more powerful, applicable to samples otherwise too small for analysis.

1) Sequencing and the detection of genetic diseases

2) Recombinant DNA techniques

3) Genetic fingerprinting and paternity testing


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

Polymerase Chain Reaction?

-The polymerase chain reaction (PCR) is a biochemistry and molecular biology technique for isolating and exponentially amplifying a fragment or sequence of interest of DNA, via enzymatic replication, without using a living organism (such as E. coli or yeast).
-As PCR is an in vitro technique, it can be performed without restrictions on the form of DNA, and it can be extensively modified to perform a wide array of genetic manipulations.

PCR principle and procedure

-PCR is used to amplify specific regions of a DNA strand.
-This can be a single gene, just a part of a gene, or a non-coding sequence.
-PCR, as currently practiced, requires several basic components
These components are:
1) DNA template that contains the region of the DNA fragment to be amplified
One or more primers, which are complementary to the DNA regions at the 5' and 3' ends of the DNA region that is to be amplified.

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

3) Deoxynucleotide triphosphates, (dNTPs) from which the DNA polymerase builds the new DNA

4) Buffer solution, which provides a suitable chemical environment for optimum activity and stability of the DNA polymerase

5) Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis

6) Monovalent cation potassium ions

The PCR is carried out in small reaction tubes (0.2-0.5 ml volumes), containing a reaction volume typically of 15-100 μl, that are inserted into a thermal cycler. This machine heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction.


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

Credits...

Sources of information for entries from June 9 to July 6:

1) http://www.csa.com/discoveryguides/gmfood/overview.php

2) en.wikipedia.org/wiki/Genetically_modified_food

3) http://www.ornl.gov/sci/techresources/Human_Genome/elsi/gmfood.shtml

4) http://www.cartoonstock.com

What is the level of detectability of GM food cross-contamination?

Scientists agree that current technology is unable to detect minute quantities of contamination, so ensuring 0% contamination using existing methodologies is not guaranteed. Yet researchers disagree on what level of contamination really is detectable, especially in highly processed food products such as vegetable oils or breakfast cereals where the vegetables used to make these products have been pooled from many different sources. A 1% threshold may already be below current levels of detectability.

How are GM foods regulated? What are government's role in this process?

Governments around the world are hard at work to establish a regulatory process to monitor the effects of and approve new varieties of GM plants. Yet depending on the political, social and economic climate within a region or country, different governments are responding in different ways.

1) Japan
The Ministry of Health and Welfare has announced that health testing of GM foods will be mandatory as of April 2001.

:. Trend:Japanese supermarkets are offering both GM foods and unmodified foods, and customers are beginning to show a strong preference for unmodified fruits and vegetables.

2)India
The government has not yet announced a policy on GM foods because no GM crops are grown in India and no products are commercially available in supermarkets yet.

:. Trend: India is very supportive of transgenic plant research. It is highly likely that India will decide that the benefits of GM foods outweigh the risks because Indian agriculture will need to adopt drastic new measures to counteract the country's endemic poverty and feed its exploding population.

3) Brazil
Some states in Brazil have banned GM crops entirely, and the Brazilian Institute for the Defense of Consumers, in collaboration with Greenpeace, has filed suit to prevent the importation of GM crops.

:. Trend: Brazilian farmers resorted to smuggling GM soybean seeds into the country because they fear economic harm if they are unable to compete in the global marketplace with other grain-exporting countries.

4) Europe
Anti-GM food protestors have been especially active. In the last few years Europe has experienced two major foods scares: bovine spongiform encephalopathy (mad cow disease) in Great Britain and dioxin-tainted foods originating from Belgium.

:. Trend: The food scares have undermined consumer confidence about the European food supply, and citizens are disinclined to trust government information about GM foods. In response to the public outcry, Europe now requires mandatory food labeling of GM foods in stores.

5) United States
The regulatory process is confused because there are three different government agencies that have jurisdiction over GM foods.

wHy?

bEcAuSe...

EPA evaluates GM plants for environmental safety, the USDA evaluates whether the plant is safe to grow, and the FDA evaluates whether the plant is safe to eat.

EPA responsible for:
Regulating substances such as pesticides or toxins that may cause harm to the environment. GM crops such as B.t. pesticide-laced corn or herbicide-tolerant crops but not foods modified for their nutritional value fall under the purview of the EPA.

USDA responsible for:
GM crops that do not fall under the umbrella of the EPA such as drought-tolerant or disease-tolerant crops, crops grown for animal feeds, or whole fruits, vegetables and grains for human consumption.

FDA:
Historically has been concerned with pharmaceuticals, cosmetics and food products and additives, not whole foods. Under current guidelines, a genetically-modified ear of corn sold at a produce stand is not regulated by the FDA because it is a whole food, but a box of cornflakes is regulated because it is a food product. The FDA's stance is that GM foods are substantially equivalent to unmodified, "natural" foods, and therefore not subject to FDA regulation.

Therefore, due to conflicts between these internal authorities, USA do not have a clear stand towards GM Food/ GMO.