1. How does the length of the recognition site determine the length of the DNA f
ID: 7707 • Letter: 1
Question
1. How does the length of the recognition site determine the length of the DNA fragments produced by the restriction enzyme (frequency of cutting)?2. Identify 3 important features of cloning vectors?
3. How are Southern Blots, Northern Blots and Western Blots similar? How are they different?
4. What are the 3 main steps to a PCR cycle and what happens during each step?
5. What are the chemical components in a PCR reaction? How are the components different in a sequencing reaction?
6. How is PCR similar to cloning? What are the advantages to PCR over cloning?
7. What are primers? How do they specify which segment of DNA is going to be amplified?
8. Could you perform whole genome sequencing from a cDNA library? Explain why or why not?
Explanation / Answer
1). Recognition site determine the length of the DNA fragments produced by the restriction enzyme. Because it is the sequenc at which the fragment is cut and DNA fragments are produced depending on the cleavage site. The frequency of restriction enzyme recognition depends on the number of nucleotides used in the site recognition. Each position of the recognition site has four possible bases and therefore the probability of finding any one base is 1/4. The length of DNA fragment will be the number of base pairs between two restriction sites.
2).Three important features of cloning vectors are
- sequences that are needed to propagate itself.
- clonong sites.
- selectable markers.
3). Southern Blots, Northern Blots and Western Blots are similar in their principle of separating the molecules. All the three techniques utilize nitrocellulose membrane and the property of complimentary and capillarity.
These three differ in the type of molecules they detect. Southern blot is used for DNA, northern blot for RNA, and Western blot for proteins.
4). 3 main steps of PCR cycle are
- Denaturation- at 94C. double strand melts open to single stranded DNA
- Annealing - at 54C. Bond betweeen primers and template occurs and polymerase reaction starts.
-Extension- 72C. Extension of the fragment takes place.
5). chemical components in a PCR reaction- DNA template, primers, Taq polymerase, dNTPs, buffer, and ions. components different in a sequencing reaction - DNA template, primers, ddNTPs, buffer, polymerase and ions.
6). PCR and cloning both involves the amplifictaion process. In pcr the DNA fragments ar eamplified, where as in cloning the production of genetically identical organisms is done. PCR is more sensitive and rapid and accurate as it is at genetic level.
7). Primers are short segments of nucleotides used to initiate the process of replication. They can specify which segment of DNA is going to be amplified due to the complimentary nature of primer with template.
8). No, whole genome sequencing cannot be done using cDNA libraries. Because only some of the sequences can be predicted where as others have to be experimentally sequenced.
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