DNA amplification: PCR
I've been able to discuss how to work with the hereditary material asacol above. Rather, as prepared to take advantage. In order to have multiple copies of a specific region of DNA, using technology Chain Reaction Polymerase (PCR = Polymerase Chain Reaction). This "great invention" asacol we owe the Nobel prize in chemistry (do not understand why they continue on without asacol giving Nobel prizes Genetics;-S) Kary B. Mullis. Came perform in vitro conditions for obtaining copies of DNA fragments. DNA is a double helix. To be able to amplify each strand, it is necessary to break the existing connections in order to maintain this structure. asacol This process is performed by raising the temperature to about 95C for a short period of time. Is called denaturation. Subsequently requires that primers (oligonucleotides or short DNA sequences of about 20 nucleotides designed for a specific area flanking DNA amplification is wanted) asacol are anchored to their complementary sequences. This temperature plays an important role, since each pair of primers (always talk about couples asacol since it must have a first or primer on one side and another on the other to be amplified the same fragment on both sides and produce amplifying the region flanked) hybridizes (binds to DNA) at a temperature that generally vary between 45 and 65 C. Although there are protocols in which temperature variations are used to get better performance, asacol but this I will discuss in later articles. Finally, we need an extension of the fragments flanked by the primers by the action of a molecule called Polymerase and having its optimum reaction temperature at 72 C (although it is also used an optimum temperature of 68 C, depending on the house supplying polymerase). Therefore, and in summary the whole process has three phases: denaturation, primer annealing and extension of the fragments. At the end of the whole program, you enter a longer extension phase to be completed to obtain a higher number of copies. This entire process is performed in the thermocycler: Reactions were prepared in the cold and the reaction tubes or plates asacol are deposited on the devices that are programmed to perform the cycles that explained above. An outline asacol of the program is to put in the picture below: In each amplification asacol cycle, the target DNA fragment increase in the copy number exponentially. This causes the end of a basic program to be obtained approximately 100 million copies of the desired fragment. Time variations depend chiefly on the fragment length. The longer the greater the fragment to be amplified. The key to a good efficiency depends on the designs of the PCR reactions to be adjusted to the conditions of each reaction. At first, the polymerases that were used were not termorresitentes. This caused, in each cycle, it was necessary to add polymerase amplification could be extended. Now polymerases are used that allow being added in the preparation asacol of the reactions and can forget about it. I would explain everything: as you design primers, performing the PCR reaction and all components, studies can be derived .. etc, but just not quite with the article and I think that I have based themselves reflected . If you have any doubts you can leave a comment or send me an email. asacol This video explains everything pretty well with English very easy to follow. Even fragments are observed which are not specific (not seek amplify but anyway obtained by hybridization of the primers) Video Reference: Essential Cell Biology, 3rd Edition. Alberts, Bray, Hopkin, Johnson, Lewis, asacol Raff, Roberts, & Walter
Related asacol Articles: AmplifX Thermocyclers wonderful thing micropipettes AmpliGrid Restriction enzymes: system on single asacol cell PCR If you enjoyed this post, make sure you subscribe to my RSS feed!
February 24, 2010 at 4:01 pm
Hello doctor, to see if I can answer this question, I searched a lot of information on CRP but find nothing. The question is: Why the amplification reaction is so specific and only amplifies a specific DNA fragment?
Hi Andrew. The answer is simpler than it seems. Specificity to band (coupled to DNA) primers or primers is enormous especially as the number of nucleotides used more than 16. I think with a numerical example is simpler: If you have to banding a single nucleotide (A, T, C or G), the probability that they will make 1/4. If you are looking for
I've been able to discuss how to work with the hereditary material asacol above. Rather, as prepared to take advantage. In order to have multiple copies of a specific region of DNA, using technology Chain Reaction Polymerase (PCR = Polymerase Chain Reaction). This "great invention" asacol we owe the Nobel prize in chemistry (do not understand why they continue on without asacol giving Nobel prizes Genetics;-S) Kary B. Mullis. Came perform in vitro conditions for obtaining copies of DNA fragments. DNA is a double helix. To be able to amplify each strand, it is necessary to break the existing connections in order to maintain this structure. asacol This process is performed by raising the temperature to about 95C for a short period of time. Is called denaturation. Subsequently requires that primers (oligonucleotides or short DNA sequences of about 20 nucleotides designed for a specific area flanking DNA amplification is wanted) asacol are anchored to their complementary sequences. This temperature plays an important role, since each pair of primers (always talk about couples asacol since it must have a first or primer on one side and another on the other to be amplified the same fragment on both sides and produce amplifying the region flanked) hybridizes (binds to DNA) at a temperature that generally vary between 45 and 65 C. Although there are protocols in which temperature variations are used to get better performance, asacol but this I will discuss in later articles. Finally, we need an extension of the fragments flanked by the primers by the action of a molecule called Polymerase and having its optimum reaction temperature at 72 C (although it is also used an optimum temperature of 68 C, depending on the house supplying polymerase). Therefore, and in summary the whole process has three phases: denaturation, primer annealing and extension of the fragments. At the end of the whole program, you enter a longer extension phase to be completed to obtain a higher number of copies. This entire process is performed in the thermocycler: Reactions were prepared in the cold and the reaction tubes or plates asacol are deposited on the devices that are programmed to perform the cycles that explained above. An outline asacol of the program is to put in the picture below: In each amplification asacol cycle, the target DNA fragment increase in the copy number exponentially. This causes the end of a basic program to be obtained approximately 100 million copies of the desired fragment. Time variations depend chiefly on the fragment length. The longer the greater the fragment to be amplified. The key to a good efficiency depends on the designs of the PCR reactions to be adjusted to the conditions of each reaction. At first, the polymerases that were used were not termorresitentes. This caused, in each cycle, it was necessary to add polymerase amplification could be extended. Now polymerases are used that allow being added in the preparation asacol of the reactions and can forget about it. I would explain everything: as you design primers, performing the PCR reaction and all components, studies can be derived .. etc, but just not quite with the article and I think that I have based themselves reflected . If you have any doubts you can leave a comment or send me an email. asacol This video explains everything pretty well with English very easy to follow. Even fragments are observed which are not specific (not seek amplify but anyway obtained by hybridization of the primers) Video Reference: Essential Cell Biology, 3rd Edition. Alberts, Bray, Hopkin, Johnson, Lewis, asacol Raff, Roberts, & Walter
Related asacol Articles: AmplifX Thermocyclers wonderful thing micropipettes AmpliGrid Restriction enzymes: system on single asacol cell PCR If you enjoyed this post, make sure you subscribe to my RSS feed!
February 24, 2010 at 4:01 pm
Hello doctor, to see if I can answer this question, I searched a lot of information on CRP but find nothing. The question is: Why the amplification reaction is so specific and only amplifies a specific DNA fragment?
Hi Andrew. The answer is simpler than it seems. Specificity to band (coupled to DNA) primers or primers is enormous especially as the number of nucleotides used more than 16. I think with a numerical example is simpler: If you have to banding a single nucleotide (A, T, C or G), the probability that they will make 1/4. If you are looking for
No comments:
Post a Comment