A.  Transformation
           1.  Purpose - To incorporate a plasmid into a bacterium's genome.  Transformation allows for different DNA to be added to a bacterial cell to supplement that already present in its circular chromosome.  This could allow for relatively large amounts of a particular gene product to be manufactured (by the bacteria) in a short period of time.

                       

           2.  Procedure:
                a)  Grow the bacteria at 37C overnight in order to produce a large number of cells.
                b)  Add plasmid DNA to the bacterial culture.
                c)  Place on ice for 15 minutes.
                d)  Heat shock at 42C for 1 minute.
                e)  Place on ice for 2 minutes.
                f)  Incubate culture at 37C for 10 minutes - 24 hours to allow for gene production.

           3.  Applications - Transformations are most often used to obtain large amounts of a particular gene product.  It is useful because the bacteria do most of the work, by actually producing the protein.  Specifically, it can be used to obtain insulin for use in diabetic patients.

           4.  Example - The following example shows how one can determine if transformation was successful.
                a)  Bacterial DNA - contains genes to metabolize glucose
                b)  Plasmid DNA - contains lac operon to metabolize lactose
                c)  Procedure:
                    1)  Take two cultures of bacteria and identify one as the experimental setup and the other as the control.
                    2)  Follow the transformation procedure outlined above for both cultures.
                    3)  However, ONLY add plasmid DNA to the experimental setup.  Take the control culture through the remainder of the steps without the plasmid DNA.
                    4)  Spread the cultures on a series of Petri dishes containing different mediums in order to determine if the transformation was successful.

                                        Glucose Plate         Lactose Plate
Experimental Setup            Growth                     Growth
Control Setup                     Growth                   No Growth

                    5)  Since the Experimental Culture was able to grow on the lactose plate, it can be confirmed that the transformation was successful.  The plasmid DNA must be present in these cells because the lactose is able to be metabolized.
                    6)  Since the Control Culture was never given any plasmid DNA, it was only able to utilize the glucose.
                    7)  This transformation appears successful.

     B.  DNA Sequencing
           1.  Purpose - To determine the linear arrangement of nucleotides in a given segment of DNA.

           2.  Procedure:
                a)  Obtain DNA segment of interest.
                b)  Make multiple copies of DNA segment.
                c)  Divide DNA into four separate test tubes.
                d)  Each test tube contains an enzyme specific for a different DNA base (Adenine, Thymine, Cytosine or Guanine).  The enzyme destroys the base that it targets, but does not harm the other three bases.
                e)  All of the A bases are removed in the Adenine test tube, and so on in the remaining three tubes for the base targeted.
                f)  The contents of each tube are loaded into separate wells of an agarose gel.
                g)  Gel electrophoresis is performed.
                h)  The banding pattern produced in the gel allows for the DNA sequence to be determined.

           3.  Applications:
                a)  Identification of Disease-Causing Genes
                b)  Disease Prevention 
                c)  Disease Correction via Genetic Engineering
                d)  Human Genome Project
                e)  Research Purposes
                f)  Numerous Ethical Issues Involved

            4.  Example of DNA Sequencing:

     C.  DNA Fingerprinting
           1.  Purpose - To compare DNA samples from various sources for similarities and differences.

           2.  Procedure:
                a)  Obtain DNA samples for testing (from crime scene or other parties involved).
                b)  Use restriction enzymes to cut DNA into smaller segments.
                c)  Incubates DNA with enzymes for approximately 60 minutes at 37C.
                d)  Load DNA samples into wells of an agarose gel.
                e)  Gel electrophoresis is performed.
                f)  Compare and contrast banding patterns produced by different DNA segments.

           3.  Applications:
                a)  Forensic (Crime Scene) Analysis
                b)  Paternity Tests

            4.  Example of DNA Fingerprinting:



** For more information on Forensic Science, please click on the link below.

                               

To test your knowledge about Recombinant DNA Applications, click on the Applications Questions Link at the top of this page.  After you answer the questions, be sure to check your responses by clicking on the Applications Answers Link.

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