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.

Controversies of GM food

This is a major concerns from Environmental activists, religious organizations, public interest groups, professional associations and other scientists and government about GM foods. Most concerns about GM foods fall into three categories: I) Environmental hazards, II) Human health risks, and III) Economic concerns.

I) Environmental hazards

1) Unintended harm to other organisms.

Case study: Last year a laboratory study was published in Nature showing that pollen from B.t. corn caused high mortality rates in monarch butterfly caterpillars. Monarch caterpillars consume milkweed plants, not corn, but the fear is that if pollen from B.t. corn is blown by the wind onto milkweed plants in neighboring fields, the caterpillars could eat the pollen and perish. Although the Nature study was not conducted under natural field conditions, the results seemed to support this viewpoint. Unfortunately, B.t. toxins kill many species of insect larvae indiscriminately; it is not possible to design a B.t. toxin that would only kill crop-damaging pests and remain harmless to all other insects. This study is being reexamined by the USDA, the U.S. Environmental Protection Agency (EPA) and other non-government research groups, and preliminary data from new studies suggests that the original study may have been flawed.

2) Reduced effectiveness of pesticides

Just as some populations of mosquitoes developed resistance to the now-banned pesticide DDT, many people are concerned that insects will become resistant to B.t. or other crops that have been genetically-modified to produce their own pesticides.

3) Gene transfer to non-target species

Another concern is that crop plants engineered for herbicide tolerance and weeds will cross-breed, resulting in the transfer of the herbicide resistance genes from the crops into the weeds. These "superweeds" would then be herbicide tolerant as well. Other introduced genes may cross over into non-modified crops planted next to GM crops.

Possible solutions to ensure that non-target species will not receive introduced genes from GM plants:

-Create GM plants that are male sterile (do not produce pollen) or to modify the GM plant so that the pollen does not contain the introduced gene. Cross-pollination would not occur, and if harmless insects such as monarch caterpillars were to eat pollen from GM plants, the caterpillars would survive.

OR

-Create buffer zones around fields of GM crops. For example, non-GM corn would be planted to surround a field of B.t. GM corn, and the non-GM corn would not be harvested. Beneficial or harmless insects would have a refuge in the non-GM corn, and insect pests could be allowed to destroy the non-GM corn and would not develop resistance to B.t. pesticides. Gene transfer to weeds and other crops would not occur because the wind-blown pollen would not travel beyond the buffer zone. Estimates of the necessary width of buffer zones range from 6 meters to 30 meters or more.
Disadvantage: Too much acreage is required for the buffer zones.

II) Human health risks

1) Allergenicity

Many children in the US and Europe have developed life-threatening allergies to peanuts and other foods. There is a possibility that introducing a gene into a plant may create a new allergen or cause an allergic reaction in susceptible individuals

2)Unknown effects on human health

There is a growing concern that introducing foreign genes into food plants may have an unexpected and negative impact on human health in the future or after a period of long term consumption.

III) Economic concerns

1) Bringing a GM food to market is a lengthy and costly process, and of course agri-biotech companies wish to ensure a profitable return on their investment. Many new plant genetic engineering technologies and GM plants have been patented, and patent infringement is a big concern of agribusiness. However, consumer advocates are worried that patenting these new plant varieties will raise the price of seeds so high that small farmers and third world countries will not be able to afford seeds for GM crops, thus widening the gap between the wealthy and the poor.

Possible way to overcome this problem:

-Introduce a "suicide gene" into GM plants. These plants would be viable for only one growing season and would produce sterile seeds that do not germinate. Farmers would need to buy a fresh supply of seeds each year.

Disadvantage of the method:

-Financial burden for farmers in third world countries who cannot afford to buy seed each year and traditionally set aside a portion of their harvest to plant in the next growing season.

The advantages of GM food

The world population has topped 6 billion and is predicted to double in the next 50 years. To ensure an adequate food supply for this booming population is going to be a major challenge in the years to come. However, GM foods may be able to meet this need in a number of ways.

:. AdvaNtaGes of GM foOds?

1) Pest resistance:

Crop losses from insect pests can result in devastating financial loss for farmers and starvation in developing countries. Farmers typically use many tons of chemical pesticides annually. However, consumers do not wish to eat food that has been treated with pesticides because of potential health hazards, and these large amount of fertilizers and pesticides used could poison the water supply and cause harm to the environment.

2) Herbicide tolerance:

For some crops, it is not cost-effective to remove weeds by physical means such as tilling, so farmers will often spray large quantities of different herbicides (weed-killer) to destroy weeds, a time-consuming and expensive process, that requires care so that the herbicide doesn't harm the crop plant or the environment. Crop plants genetically-engineered to be resistant to one very powerful herbicide could help prevent environmental damage by reducing the amount of herbicides needed.
Disease resistance There are many viruses, fungi and bacteria that cause plant diseases. Plant biologists are working to create plants with genetically-engineered resistance to these diseases.

3) Cold tolerance:

Unexpected frost can destroy sensitive seedlings. An antifreeze gene from coldwater fish has been introduced into plants such as tobacco and potato. With this antifreeze gene, these plants are able to tolerate cold temperatures that normally would kill unmodified seedlings

4) Drought tolerance/salinity tolerance:

As the world population grows and more land is utilized for housing instead of food production, farmers will need to grow crops in locations previously unsuited for plant cultivation. Creating plants that can withstand long periods of drought or high salt content in soil and groundwater will help people to grow crops in formerly inhospitable places

5) Nutrition:

Malnutrition is common in third world countries where impoverished peoples rely on a single crop such as rice for the main staple of their diet. However, rice does not contain adequate amounts of all necessary nutrients to prevent malnutrition. If rice could be genetically engineered to contain additional vitamins and minerals, nutrient deficiencies could be alleviated. For example, blindness due to vitamin A deficiency is a common problem in third world countries. Researchers at the Swiss Federal Institute of Technology Institute for Plant Sciences have created a strain of "golden" rice containing an unusually high content of beta-carotene (vitamin A). Since this rice was funded by the Rockefeller Foundation, a non-profit organization, the Institute hopes to offer the golden rice seed free to any third world country that requests it.

6) Pharmaceuticals:

Medicines and vaccines often are costly to produce and sometimes require special storage conditions not readily available in third world countries. Researchers are working to develop edible vaccines in tomatoes and potatoes. These vaccines will be much easier to ship, store and administer than traditional injectable vaccines.