1 a. The source organism for Taq DNA polymerase is a thermophillic bacterium, Thermus aquaticus.
In PCR, the DNA double strand is denatured and this is generally done at around 72-75oC depending upon the GC content of the DNA template. More over protein contamination can affect DNA amplification. Hence to denature the protein, higher temperature is used to denature the DNA along with the protein contamination. This higher temperature can’t be tolerated by the normal DNA polymerase. Taq polymerase has a half life of around two hours at 93oC and can replicate the DNA at the rate of 1000bp/sec at 72oC. Hence Taq pol is used in PCR.
b) Template DNA can be extracted with an intermediate step in which protein is denatured before PCR. This can reduce the need of higher temperature for protein denaturation. We can also use other methods to denature DNA like using low salt concentration or alkaline pH in the reaction mixture. We can also increase time of denaturation and carry on the reaction at a lower and optimum temperature for the normal DNA polymerase enzyme. Simply the best method will be do the individual steps in individual vials stepwise instead of doing it in an automated PCR machine.
2 a. Restriction endonucleases are the enzymes that cut the DNA at particular sites. Because of the discovery of this enzyme, molecular biologists were able to play with the DNA. Analysing the long DNA sequence as a whole was very difficult. But cutting the DNA into small fragments and analysing the individual fragments separately was quite easier. These DNA fragments can be inserted anywhere, like in plasmids or any vectors and then anything can be done with the fragments. Whether it may be expressing certain gene to get a particular protein, or it may be sequencing the whole gene or the genome; many things could be done with it. This started a new era of molecular biology.
b. Generally these restriction enzymes are present in the bacteria to avoid bacteriophage attack. When the phage invades the bacteria, it casts its DNA into the bacterial cell. If the bacterial cell has the restriction enzymes in it, then they cut the viral genetic material arbitrarily at any point. This renders the Viral DNA inactive and it cannot carry out its normal life cycle in the bacterial host system.
Generally the cell produces certain enzymes, like the methylase. These enzymes epigenetically modify the host DNA at the recognition sequences of the restriction enzymes by methylating the adenine bases. As a result of this, the endonucleases are not able to act on these methylated hosts DNA and the host DNA is not affected. (NCBI, NBK22528)
1 A的源生物的Taq DNA聚合酶是一种嗜热菌Thermus aquaticus中。
在PCR，DNA双链变性，这通常是在72-75oc取决于模板DNA的GC含量做了。以上蛋白污染可以影响DNA扩增。从而使蛋白质变性，使用更高的温度使DNA变性随着蛋白质污染。这种更高的温度不能用正常的DNA聚合酶的耐受性。Taq DNA聚合酶具有在93oc两小时半的生活，可以复制的DNA在1000bp /秒的速度72oc。因此，用PCR Taq聚合酶。