This is a study guide for a final that I\'ve already looked thorugh and answered

Question

This is a study guide for a final that I've already looked thorugh and answered but wanted to compare my answers to others in case I missed information or the point of the question. Cheers! Thanks a bunch for you help!!

Explain how each of these technologies is involved in cloning technologies: restriction enzymes, bacterial plasmids, gel electrophoresis, and transformation.

Diagram a plasmid vector and all the desirable features you would want in a plasmid cloning vector. Explain the utility of each feature.

What is meant by insert DNA? What are potential sources for insert DNA?

What is included in a cloning ligation reaction? What are the potential products? How will those products behave in a host cell?

What is the difference between screening and selection? What is the difference between recombinant and non-recombinant plasmids?

What manipulations are done, and why, to improve transformation frequency when using the chemical transformation method? Compare chemical transformation to electroporation.

What features would you want in a host cell for bacterial transformation and why?

Outline, in brief, the alkaline-lysis mini-prep procedure, how do the steps allow the isolation of plasmid DNA relatively free of other host cell components?

Why are TOPO vectors used for cloning PCR products? What is the efficiency of TOPO cloning over ligation?

How are Lambda () viruses modified to make useful cloning vectors? Describe how cloning vectors and insert DNA are prepared to carry out cloning. What advantages do -based vectors have over plasmid vectors?

Compare plasmid and -based vectors to cosmid vectors, what are the advantages of cosmid vectors? What features would you expect to find in a Yeast Artificial Chromosome, what is their purpose?

What is the advantage of BACs over YACs?

Give the approximate insert size accepted by the various cloning vectors?

What are the various types of cloning libraries? What is the advantage/use of each type? How is the source material prepared for each type of library?

What variables determine the number of clones needed to get reasonable coverage in a genomic library?

In screening various libraries by colony or plaque hybridization what are some likely sources for your probes?

Describe how differential screening can be used to find cell type specific clones. How is chromosome walking used to locate a gene sequence?

What is an ordered library? How can it be efficiently screened?

Describe how transposon tagging can be used to find/clone a gene. What is rescue cloning in relation to transposon tagging?

Explanation / Answer

DNA cloning is the process of making multiple, identical copies of a particular piece of DNA.

Plasmids in Cloning- In a typical DNA cloning procedure, the gene or other DNA fragment of interest (perhaps a gene for a medically important human protein) is first inserted into a circular piece of DNA called a plasmid. The insertion is done using enzymes that “cut and paste” DNA, and it produces a molecule of recombinant DNA, or DNA assembled out of fragments from multiple sources. Next, the recombinant plasmid is introduced into bacteria. Bacteria carrying the plasmid are selected and grown up. As they reproduce, they replicate the plasmid and pass it on to their offspring, making copies of the DNA it contains.

Restriction enzymes in cloning - Restriction enzymes are found in bacteria (and other prokaryotes). They recognize and bind to specific sequences of DNA, called restriction sites. Each restriction enzyme recognizes just one or a few restriction sites. When it finds its target sequence, a restriction enzyme will make a double-stranded cut in the DNA molecule. Typically, the cut is at or near the restriction site and occurs in a tidy, predictable pattern. In DNA cloning, restriction enzymes and DNA ligase are used to insert genes and other pieces of DNA into plasmids.

Gel Electrophoresis in cloning - Gel electrophoresis separates DNA molecules of different sizes.  For DNA fragments less than 500 nucleotides long, specially designed polyacrylamide gels allow separation of molecules that differ in length by as little as a single nucleotide. The pores in polyacrylamide gels, however, are too small to permit very large DNA molecules to pass; to separate these by size, the much more porous gels formed by dilute solutions of agarose (a polysaccharide isolated from seaweed) are used. These DNA separation methods are widely used for both analytical and preparative purposes. Analytical purposes usually involve a blotting and hybridization step following the gel electrophoresis. This enables the researcher to identify the DNA and verify the veracity of the cloning experiment.

Transformation in cloning - In a typical cloning experiment, researchers first insert a piece of DNA, such as a gene, into a circular piece of DNA called a plasmid. This step uses restriction enzymes and DNA ligase and is called a ligation. After a ligation, the next step is to transfer the DNA into bacteria in a process called transformation. Then, we can use antibiotic selection and DNA analysis methods to identify bacteria that contain the plasmid we’re looking for.

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