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9.6 Option – Biotechnology: 5. Recombinant DNA
Extract from Biology Stage 6 Syllabus (Amended October 2002). © Board of Studies, NSW.
Prior learning: Stage 4-5 Syllabus,
5.8.2(a, b, c and d).
H.S.C. module 9.3 (subsections 3 and 5).
Background information: The term recombinant DNA refers to DNA that has been cut from one strand of DNA and then inserted into the gap of another piece of DNA that has been broken. The host DNA is often a bacterial cell such as E coli. The purpose of splicing the DNA, eg human DNA into the host DNA is to produce many copies of it. As bacteria reproduce in a very short time it is possible to make millions of copies of the gene fairly quickly. Rather than using scissors to cut and glue to splice or paste, enzymes play this very important role. Recombinant DNA has a very wide application such as human health, eg producing human insulin outside the body for diabetics, food production, eg cows that have more milk or a plant that is resistant to a pest so that spraying for that pest can be avoided. Mapping of DNA had to be done before this process became possible.
describe the three essentials of gene manipulation as:
- cutting and joining DNA
- monitoring the cutting and joining
- transforming hosts, such as bacteria, with the recombinant DNA
Cutting and Joining
- The DNA that is required is cut from a long strand of DNA (a chromosome), using enzymes. This is actually a long, complex process but great detail is not required here. An example could be the gene for making human insulin is cut from a human pancreas cell using a restriction enzyme.
- A circular piece of DNA, called a plasmid, that is present in bacterial cells, is removed from the cells and is cut open using the same restriction enzyme.
- The cut out human gene is then mixed with the bacterial plasmids in a test tube. Because they have been cut with the same enzyme, the cut ends of the plasmid and the end of the human gene match.
- The enzyme DNA ligase is used to stick the ends together.
A series of
diagrams illustrating cutting and joining 
, College of St
Benedict/ St John’s University, St Cloud, Minnesota,
USA.
Monitoring
- This has to be done to make sure the correct gene is spliced into the plasmid. Unwanted DNA fragments have to be removed. This is done by electrophoresis.
- All the DNA are placed in depressions at one end of a slab of agar gel. Electrodes are placed at each end of the gel to create an electric field.
- Because the DNA molecules are negatively charged, the DNA fragments travel through the gel towards the positive end. Since smaller pieces move faster than larger ones, the various fragments are separated by size and the required ones can be easily obtained. These correct genes are placed into the plasmids as described above.
- It is necessary to isolate the host bacteria that contain the gene that has been spliced as you don’t want the bacteria that don’t contain this gene. By having a gene on the same plasmid that gives resistance to an antibiotic, the other bacteria can be removed by culturing the bacteria in a medium that contains the antibiotic. The bacteria containing the resistance to the antibiotic will survive and the others will be killed by the antibiotic.
Another
good diagram , Access Excellence, National Health Museum,
USA. This diagram shows the gene for resistance to the
antibiotic in the plasmid.
Transforming hosts
- This refers to replacing the plasmids that contains the introduced gene (recombinant DNA) into the E coli bacteria so they can multiply and make more of the gene. It can be done by combining them in a test tube with calcium chloride. The high concentration of calcium ions makes the membranes of the bacteria more porous. This then allows the plasmids to move into the bacterial cells. Not all bacteria will take up a plasmid and this is why the monitoring described above must happen.
process
information to produce a flow chart on the sequence of
events that result in the formation of recombinant
DNA
- Use the information from the dot point above and add to this information if you think it necessary by looking in books, the Internet and journals.
-
A good site to start with , John W Kimball, USA.
- If you have used several sources you will need to process the information by organising all of it in a sequence. Take the main points and summarise them in sentences or in labelled diagrams. Put these ordered summaries in boxes then join them with arrows so they are in the form of a flow chart . You could put this flow chart on cardboard to display to your teacher or to other students or it could be made into a power point display.
perform a
first-hand investigation to extract and identify DNA
from a suitable source
- Your teacher may suggest a method of extracting DNA or
you could find a method on the Internet.
Extracting DNA in your kitchen Scientists and Discovery, Museum Victoria. Another good site is
Extracting DNA , NCBE, Reading, UK.
- Make sure you have the necessary equipment for the investigation and be sure you carry out any safety precautions given.
- Write up your investigation, stating how successful you were.
gather and
analyse
information to outline the
purpose of a current application of transgenic technology,
naming the organism and gene transfer technique
involved
- As you have to find a current application of transgenic technology, it is probably best to gather information on the Internet. Use a search engine such as Google and type in key words. You could start with ‘transgenic’, ‘current’, ‘gene tranfer’. You might like to read a few examples before you decide which application you are going to analyse.
Note: Be careful that the articles you find are up to date by checking for a date on the article. To be current it shouldn’t be more than one or two years old.
- When analysing the information you should draw out and relate implications of the issues being considered. There are many examples of transgenic technologies so choose one that is of interest to you. It may involve plants that are used for food or it may involve an aspect of an animal that is a food such as the muscle of an animal that is used for meat. You could consider advantages and disadvantages but be aware that ethical issues are considered in the last dot point of the option.
- Note that you have to outline the purpose of a current application of transgenic technology, naming the organism and gene transfer technique involved. Outline only involves sketching in general terms and indicating the main features of a technology so don’t go into more detail than is required.
describe the following recombinant DNA techniques used in biotechnology, including:
- gene splicing using restriction enzymes and ligases to produce recombinant DNA
- polymerase chain reaction to amplify or modify DNA sequences
- use of DNA vectors and microinjection for carrying genes into nuclear DNA in the production of transgenic multicellular organisms
Gene splicing using restriction enzymes and ligases to produce recombinant DNA
- The gene for insulin production in humans can be pasted into the DNA of Escherichia coli, a bacterium that inhabits the human digestive tract. This is done by cutting the appropriate gene from human DNA and pasting, or splicing, it into plasmid DNA, a special kind of DNA that takes a circular form and can be used as a vehicle for this editing job.
- It is cut using a class of enzymes called restriction enzymes. There are well over a hundred restriction enzymes, each cutting in a very precise way a specific base sequence of the DNA molecule. With these scissors used singly or in various combinations, the segment of the human DNA molecule that specifies insulin production can be isolated.
- This segment is "glued" into place using an enzyme called DNA ligase. The result is an edited, or recombinant, DNA molecule. The bacterial cells divide very rapidly making billions of copies of themselves, and each bacterium carries in its DNA a faithful replica of the gene for insulin production. Each new E. coli cell has inherited the human insulin gene sentence.
Polymerase chain reaction to amplify or modify DNA sequences
- This is by far the most successful method of amplifying (making many copies of) DNA sequences.
- The process is done in a test tube: the DNA required is isolated, fragmented with restriction enzymes and separated by gel electrophoresis. The DNA fragments are denatured (ie, made single stranded) by heating to about 95 degrees C. These single stranded DNA are exposed to a solution containing a radioactive DNA “probe”. The probe consists of single stranded DNA or RNA, with a sequence chosen to base pair with the required DNA. With correct temperature of about 50 – 65 degrees C, salt and correct pH the probe will bind to its corresponding sequence of the target DNA and nowhere else.
- As the process proceeds the DNA doubles after each cycle. Following thirty such cycles a theoretical one billion copies of DNA can be produced.
- PCR had been used to indicate the presence of HIV infection and has been used to amplify degraded DNA for use in forensic science.
Use of DNA vectors and microinjection for carrying genes into nuclear DNA in the production of transgenic multicellular organisms
- Commonly used vectors (carriers) are viruses or plasmids. A viral vector is first modified so that it will not replicate or cause disease in the target cells of the host embryo. The gene of interest is incorporated into the viral genome (See gene splicing above) and the virus is then used to infect an early stage embryo or a pronuclear embryo. The viral vector binds uniformly to the embryonic cells and acts as a vehicle to allow transfer and integration of the transgene into the host genome. If a pronuclear embryo is used the new gene will be expressed in every cell. However if the virus infects a cell of an early stage embryo not all cells will contain the new gene.
- Retroviruses are commonly used as vectors because of their ability to infect host cells in this way.
- If a retrovirus isn’t used, eg a plasmid or other virus is used it can be inserted into the cell using microinjection.
- An advantage to using viral vectors is that usually only a single copy of the transgene is integrated into the genome. If the viral transfection is applied to oocytes (eggs) prior to fertilization, then the new gene will be present in all cells of the resulting embryo as though it had been contributed by the mother.
- The major disadvantage of this system is the time and labour-intensive process to prepare the viral vector. There is also a remote possibility that the modified viral vector may revert to its original state or recombine with other pathogenic viruses. Microinjection doesn’t always result in the gene being incorporated into a chromosome in a way that it can be expressed.
process and
analyse
secondary information to identify that
complementary DNA is produced by reverse transcribing RNA or
the polymerase chain reaction
- Use the information that you gathered in the above dot point on polymerase chain reaction.
- Gather any other information you might want to add to the information you already have by looking in biology and biotechnology texts or journals.
- Process the information by extracting the parts that are relevant.
- Identify that complementary DNA is produced by reverse transcribing RNA.
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