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Gene Cloning, Manipulation, & Applications

Hw 13.
1. What is meant by the term "clone"?
Why is it important to obtain large quantities of a particular DNA sequence?
List the steps you would take to obtain a clone of a DNA sequence.

2. For each of the following, give a brief description:
restriction enzyme
vector
recombinant DNA molecule
sticky ends

3. What is the process of transformation?
What is meant by competent cells?
What would you use a selection system for?

4. What information could you obtain from a southern blot?
What information could you obtain from a northern blot?
Describe what a genomic library is.

5. You are a forensic scientist and you perform a southern analysis to try and link a possible suspect to the scene of a crime. Draw the results of such a southern analysis and decide if the suspect was present or not.
Describe a possible application for making a transgenic animal or plant.

Solution Preview

1. What is meant by the term "clone"? Why is it important to obtain large quantities of a particular DNA sequence?
List the steps you would take to obtain a clone of a DNA sequence.

The term cloning in biology has two connotations. It can be used to describe the process of making a genetic double using genomic DNA from a fully differentiated cell (think about Dolly the sheep). But the more common usage of cloning refers to making a copy of a piece of DNA to facilitate manipulating it. In order to study the function of a gene researchers will clone the gene or, more commonly, the cDNA copy of the gene. cDNA, or complimentary DNA, is a copy in DNA form of the coding region of a gene (without introns). Once the research has a copy of the coding region of a gene they can insert it into a bacterial vector and produce large copies of it in bacteria. This allows researchers to perform various experiments. For example, the cDNA can be fused to a fluorescent protein and expressed in cells to determine its sub-cellular localization. It can be inserted into a specialized vector to use in a yeast two-hybrid experiment to identify potential binding partners. One of the simplest things that can be done with this cDNA is to use it as a template to make a complimentary digoxigenin-labeled probe for in situ hybridization experiments. This technique allow researchers to determine where and when the gene is being expressed in the organism it came from. All of these experiments require a pool of the cDNA for the gene of interests to start from. Cloning that cDNA and amplifying it in bacteria is how researchers keep the cDNA on hand for manipulation.

To clone a cDNA via PCR:
-obtain a source of mRNA likely to contain the mRNA from your gene of interest. For example if your gene is expressed in the eye you would extract mRNA from an eye.
-create primers for your mRNA of choice using sequence available at GenBank. Often primers have restriction enzyme sites added to their sequence to facilitate inserting your desired sequence into a bacterial vector but it can be done without. If your sequence is not available at Gen Bank because you are using a novel organism you can design primers based on the consensus sequence of related organisms.
-reverse transcript your mRNA pool to create cDNA.
-PCR amplify your cDNA of interest from ...

Solution Summary

Short answers with web references for given questions.

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