detection of gm crops gene
this research is what i found..i'm still figuring out how it exactly works but feel free to try to understand the logic behind these.
____________________________________________________
Detecting Genetically Modified Plants
Once the plants have grown in soil, they must be tested to determine if they contain a transferred gene. Although all of the grown plants are known to contain T-DNA from between the boundary regions of the vector through antibiotic selection, it is not yet known if the gene inserted into the vector during the recombinant step or not. The main method used to determine this is a fundamental procedure of biochemistry: polymerase chain reaction (PCR). PCR amplifies specific DNA sequences, creating millions of identical molecules using just one as a template.
PCR goes through many cycles of reactions, with each cycle containing three steps. First, the double-stranded DNA molecule separates into two strands when incubated at high temperatures (about 94° C), creating two complementary single-stranded DNA molecules. Next, the temperature is dropped to 55° C and two primers complementary to specific sequences within the DNA molecule bind. The primers are DNA oligonucleotides, short stretches of single-stranded DNA, which bind sequences that precede the 5' end of the region to be amplified on either strand. These primers bind to the complementary bases on the target DNA molecule, creating short double-stranded portions of DNA. Fianlly, the temperature is raised to 75° C, at which Taq polymerase, a heat-stable DNA polymerase isolated from Thermus aquaticus, catalyzes the extension of the primers on each strand. The polymerase recognizes double-stranded DNA and adds free nucleotides (dNTPs) to the 3' ends of the regions where the primers are bound (Voet 2001).
After the first cycle, one DNA molecule has become two molecules, since each separated strand has been bound by primer and extended by the polymerase. However, while these molecules contain the region of interest, the stretch between the two primers, they contain bases past the boundaries marked by the primers, since the polymerase does not know where the other primer is and extends the chain indefinitely. A second cycle of PCR amplifies the two newly synthesized strands to produce two more molecules. These molecules still have one strand that is longer than the target molecule. After the third cycle, there are eight molecules, two of which are identical to the target. Typically, PCR is carried out in 20 or more cycles, approximately doubling the amount of DNA each time, resulting in about 220 (approx. 1 million) copies of the target molecule (Gachet 1999). PCR can be used to determine if a plant contains the desired transgene by extracting the plant's DNA and amplifying it. Since the sequence of the transgene is known and present only in transformed cells, PCR primers that recognize regions within the inserted T-DNA can be made (Gachet 1999). PCR is run on the extracted plant DNA with these primers, and a procedure called gel electrophoresis can determine its success.
In gel electrophoresis, DNA or protein samples are loaded onto a porous gel containing a network of agarose or polyacrylamide molecules. Electrodes are connected across the gel (often the gel is placed in a tank that has electrodes at its ends) causing a current to pass through it. DNA carries a negative charge, so it moves through the gel toward the positive electrode. The DNA encounters agarose or polyacrylamide molecules that hinder its path, with smaller molecules getting past these obstacles more quickly than larger ones. Since many molecules are placed onto the gel, they will separate according to size and form bands. The distances they travel are proportional to their molecular weights. The resulting separated bands can be detected using staining techniques or through the use of UV light that illuminates a fluorescent dye molecule bound to the samples before they are run.
The visualizing methods have sensitivities on the order of one nanogram (Voet 2001), so only bands containing a large number of DNA molecules can be seen. If the genes were successfully transferred to the plant, the plant DNA would contain primer recognition sites and be amplified by PCR, producing enough molecules to be seen on the gel. If the gene is not present, the primers would not bind, and only one molecule would be in the sample, producing no band when stained because of the sensitivity of the procedure. Appropriate controls -- performing PCR on a molecule that the primers should not recognize as well as one of known sequence that they should recognize -- must be used to show that known sequences were amplified and visualized only if they contained regions corresponding to the primers (Quist 2001).
Detection of transgenic DNA in wild plants is a little more complicated. Since this requires detecting DNA of unknown sequence, many different primer targets must be chosen to give the best chance of amplifying a transgene, if present. Three categories of sequences are often targeted: regulatory sequences from transforming vectors, genetic markers used to select the transformed cells during the engineering process, and sequences within the transgenes themselves (Gachet 1999). One such primer target is the P35S promoter sequence of cauliflower mosaic virus, as it is found in many commercial transgenic constructs (Quist 2001).
source:
http://www.jyi.org/volumes/volume5/issue6/features/pattanayak.html
____________________________________________________
Detecting Genetically Modified Plants
Once the plants have grown in soil, they must be tested to determine if they contain a transferred gene. Although all of the grown plants are known to contain T-DNA from between the boundary regions of the vector through antibiotic selection, it is not yet known if the gene inserted into the vector during the recombinant step or not. The main method used to determine this is a fundamental procedure of biochemistry: polymerase chain reaction (PCR). PCR amplifies specific DNA sequences, creating millions of identical molecules using just one as a template.
PCR goes through many cycles of reactions, with each cycle containing three steps. First, the double-stranded DNA molecule separates into two strands when incubated at high temperatures (about 94° C), creating two complementary single-stranded DNA molecules. Next, the temperature is dropped to 55° C and two primers complementary to specific sequences within the DNA molecule bind. The primers are DNA oligonucleotides, short stretches of single-stranded DNA, which bind sequences that precede the 5' end of the region to be amplified on either strand. These primers bind to the complementary bases on the target DNA molecule, creating short double-stranded portions of DNA. Fianlly, the temperature is raised to 75° C, at which Taq polymerase, a heat-stable DNA polymerase isolated from Thermus aquaticus, catalyzes the extension of the primers on each strand. The polymerase recognizes double-stranded DNA and adds free nucleotides (dNTPs) to the 3' ends of the regions where the primers are bound (Voet 2001).
After the first cycle, one DNA molecule has become two molecules, since each separated strand has been bound by primer and extended by the polymerase. However, while these molecules contain the region of interest, the stretch between the two primers, they contain bases past the boundaries marked by the primers, since the polymerase does not know where the other primer is and extends the chain indefinitely. A second cycle of PCR amplifies the two newly synthesized strands to produce two more molecules. These molecules still have one strand that is longer than the target molecule. After the third cycle, there are eight molecules, two of which are identical to the target. Typically, PCR is carried out in 20 or more cycles, approximately doubling the amount of DNA each time, resulting in about 220 (approx. 1 million) copies of the target molecule (Gachet 1999). PCR can be used to determine if a plant contains the desired transgene by extracting the plant's DNA and amplifying it. Since the sequence of the transgene is known and present only in transformed cells, PCR primers that recognize regions within the inserted T-DNA can be made (Gachet 1999). PCR is run on the extracted plant DNA with these primers, and a procedure called gel electrophoresis can determine its success.
In gel electrophoresis, DNA or protein samples are loaded onto a porous gel containing a network of agarose or polyacrylamide molecules. Electrodes are connected across the gel (often the gel is placed in a tank that has electrodes at its ends) causing a current to pass through it. DNA carries a negative charge, so it moves through the gel toward the positive electrode. The DNA encounters agarose or polyacrylamide molecules that hinder its path, with smaller molecules getting past these obstacles more quickly than larger ones. Since many molecules are placed onto the gel, they will separate according to size and form bands. The distances they travel are proportional to their molecular weights. The resulting separated bands can be detected using staining techniques or through the use of UV light that illuminates a fluorescent dye molecule bound to the samples before they are run.
The visualizing methods have sensitivities on the order of one nanogram (Voet 2001), so only bands containing a large number of DNA molecules can be seen. If the genes were successfully transferred to the plant, the plant DNA would contain primer recognition sites and be amplified by PCR, producing enough molecules to be seen on the gel. If the gene is not present, the primers would not bind, and only one molecule would be in the sample, producing no band when stained because of the sensitivity of the procedure. Appropriate controls -- performing PCR on a molecule that the primers should not recognize as well as one of known sequence that they should recognize -- must be used to show that known sequences were amplified and visualized only if they contained regions corresponding to the primers (Quist 2001).
Detection of transgenic DNA in wild plants is a little more complicated. Since this requires detecting DNA of unknown sequence, many different primer targets must be chosen to give the best chance of amplifying a transgene, if present. Three categories of sequences are often targeted: regulatory sequences from transforming vectors, genetic markers used to select the transformed cells during the engineering process, and sequences within the transgenes themselves (Gachet 1999). One such primer target is the P35S promoter sequence of cauliflower mosaic virus, as it is found in many commercial transgenic constructs (Quist 2001).
source:
http://www.jyi.org/volumes/volume5/issue6/features/pattanayak.html
posted by .:: Estelle ::. @ 16:09
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