Questions on DNA recombinant DNA technology?

[sameeralord]

Hello everyone,

I'm not very good at this area, so I tried to do some questions to get better, but I have many confusions as a result. This is not homework, I hope you can help me. Thank you

These are true, false questions.

*Restriction fragment length polymorphism can be used to carry out prenatal diagnosis?
I understand the concept, so I think this is true, if you use fetal tissues.
*Gel electrophoresis can be used to measure the length of the DNA accurately?
I can see how if you have standard fragment of known size you can do this but how can the distance of a fragment exactly give the size of the molecule. In electrophoresis particles with different sizes can end up at the same place due to other factors like charge and resistance. So is this false.
*Genetic engineering does not theoretically provide a care for enzyme deficiency?
This is false right
*DNA polymerase is denatured when enzymatic extension takes place at 72 degrees celsius?
I know Taq DNA polymerase is used so it wouldn't denature like dna polymerase. But I'm not sure if normal DNA polymerase denatures at 72 or 90 when melting of dna takes place?
*Isolated DNA can be rapidly sequenced by either chemical method or enzymatic method?
Not sure
*Blotting and hybridization techniques are important in the identification of the specific DNA and RNA fragment?
Here is probing considered a form of hybridization. I think this is faily true, but I don't think blotting can use RNA. Can it
*Polyacrilamide gel are used to separate DNA fragements with more than 500 nucleotides?
polyacrylamide gels to separate DNA fragments differing by a single base-pair in length , that is what wiki says. But I still don't get the answer.

Thanks

 

[Ygggdrasil]

1) You are correct, this is true.

2) You are correct that electrophoresis separates particles on factors other than size. However, these other factors, such as charge and resistance, also depend on the length of the DNA molecule. For example, the charge of the DNA molecule scales linearly with its length. Therefore, gel electrophoresis can be used to accurately measure the length of a DNA fragment.

3) In theory genetic engineering could cure an enzyme deficiency (provided the enzyme deficiency is due to a genetic disorder). In practice, this is very difficult.

4) Normal DNA polymerases would denature at the high temperatures used in a PCR reaction. Taq DNA polymerase, isolated from a thermophilic hot springs bacterium, however, does not denature at these high temperatures.

5) I'd say this is true, though might depend on one's definition of rapid.

6) Yes, blotting can be used to identify RNAs. Blotting to probe for specific DNA sequences is a technique called Southern blotting as it was invented by a man named Edwin Southern. The names of blotting methods invented subsequently play on the geographical naming theme. Blotting to probe for specific RNA sequences (a common technique to see whether a cell is expressing a certain mRNA) is called Northern blotting. A blot that detects specific proteins is called a Western blot.

7) There are two main substrates used for gel electrophoresis: agarose and polyacrylamide. Polyacrylamide gels have much smaller pore sizes than agarose gels and therefore provide higher resolution (able to separate strands differing by one base pair). The small pore size, however, means that polyacrylamide gels are unable to separate very large fragments of DNA. Agarose gel electrophoresis is much better suited for separation of DNA fragments greater than 500bp.

 

[sameeralord]

1) You are correct, this is true.

2) You are correct that electrophoresis separates particles on factors other than size. However, these other factors, such as charge and resistance, also depend on the length of the DNA molecule. For example, the charge of the DNA molecule scales linearly with its length. Therefore, gel electrophoresis can be used to accurately measure the length of a DNA fragment.

3) In theory genetic engineering could cure an enzyme deficiency (provided the enzyme deficiency is due to a genetic disorder). In practice, this is very difficult.

4) Normal DNA polymerases would denature at the high temperatures used in a PCR reaction. Taq DNA polymerase, isolated from a thermophilic hot springs bacterium, however, does not denature at these high temperatures.

5) I'd say this is true, though might depend on one's definition of rapid.

6) Yes, blotting can be used to identify RNAs. Blotting to probe for specific DNA sequences is a technique called Southern blotting as it was invented by a man named Edwin Southern. The names of blotting methods invented subsequently play on the geographical naming theme. Blotting to probe for specific RNA sequences (a common technique to see whether a cell is expressing a certain mRNA) is called Northern blotting. A blot that detects specific proteins is called a Western blot.

7) There are two main substrates used for gel electrophoresis: agarose and polyacrylamide. Polyacrylamide gels have much smaller pore sizes than agarose gels and therefore provide higher resolution (able to separate strands differing by one base pair). The small pore size, however, means that polyacrylamide gels are unable to separate very large fragments of DNA. Agarose gel electrophoresis is much better suited for separation of DNA fragments greater than 500bp.

Thanks Ygggdrasil this seems bread and butter for you Just a few questions. In question 4 regarding PCR,enzymatic extension occurs at 72 degress celsius, and the denaturation which occurs before that occurs at 90. Does normal DNA polymerase denature around both these temperature, or can it withstand 72. I just want to know which stage denaurtion occurs . In question 2, does longer DNA fragments have different charge, I thought all DNA have negative charge, does negative charge increase with increase of length due to more phosphates.

 

[Ygggdrasil]

Thanks Ygggdrasil this seems bread and butter for you Just a few questions. In question 4 regarding PCR,enzymatic extension occurs at 72 degress celsius, and the denaturation which occurs before that occurs at 90. Does normal DNA polymerase denature around both these temperature, or can it withstand 72. I just want to know which stage denaurtion occurs.

Unlike Taq polymerase, which operates best at around 72^oC, most DNA polymerases (such as those found in humans or E. coli) work best at 37^oC (human body temperature, their natural environment). I'm not sure the exact temperature that they denature, but would guess that it is somewhere in the range of 50-70^oC. If you want to kill the activity of an enzyme in molecular biology, a standard procedure is to heat the enzyme at ~65^oC for 10-30 min. This will denature most, but not all, enzymes.

In question 2, does longer DNA fragments have different charge, I thought all DNA have negative charge, does negative charge increase with increase of length due to more phosphates.

Yes, longer DNA molecules have more negative charge because they have more phosphates.