Restriction Enzymes Gel Electrophoresis Southern Blot technique Polymerase Chain Reaction DNA Sequencing

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1 Molecular Genetics

2 Restriction Enzymes Gel Electrophoresis Southern Blot technique Polymerase Chain Reaction DNA Sequencing Solving Crime Determining Paternity Gene Cloning

3 Restriction enzymes are used to cut pieces of DNA. They have specific recognition sequences where they cut. They can produce either sticky ends or blunt ends. They can be used to insert pieces of foreign DNA into bacteria or virus. The bacterial DNA and the gene to be inserted in the bacteria are cut with the same restriction enzyme, to ensure their ends match.

4 Restriction enzymes are naturally occurring proteins often found in bacteria. They are named after the bacteria and numbered in order of their discovery. For example: EcoRI the first restriction enzyme isolated from the bacteria Escherichia coli AluI the first restriction enzyme isolated from the bacteria Arthrobacter luteus

5 DNA ligase is used to join fragments of DNA together. Once DNA has been cut by a restriction enzyme and the new piece of DNA is ready to insert, DNA ligase is used to join the nucleotides together. DNA ligase occurs naturally in all organisms and is used to zip up the DNA strands after they have been copied in natural protein synthesis.

6 This is a method for separating DNA fragments according to size. The DNA sample is put into wells in a gel. The gel floats in a buffer solution and a current is passed through the solution. The DNA fragments, being slightly negative in charge moves toward the positive end of the gel. Smaller fragments move faster through the gel than larger ones, with fragments of the same size being aligned as shown in the diagram below.

7 This techniques is named for its inventor Edward Southern. It is used when one needs to work with a large sample of DNA, for example the total genome of an individual. This technique is used to determine the specific location of a specific gene.

8 Step 1. Cut the total genome with restriction enzymes. Step 2. Pick up the smear of fragments with a nitrocellulose filter paper. Step 3. Use a probe to find a particular piece of DNA of interest. Step 4. After the probe has attached to the required piece of DNA use radioactive or flourescent dye labelling to detect it.

9 This is an enzyme used to make copy DNA (single stranded DNA) from messenger RNA. Often mrna can be obtained, which contains no introns, only the coding regions of a gene. This is particularly useful for inserting genes into other organisms. Many organisms will only accept small amounts of DNA. A Poly A tail is added to the mrna, this then acts as the region for the primer to bind to. Reverse transcriptase, a naturally occurring enzyme, catalyses the addition of new bases to the cdna. Once this is made, the mrna is removed and DNA polymerase is added to build the complementary strand of DNA. The result is double stranded DNA created from the mrna base.

10 PCR is a method used to amplify DNA (make many copies of it). Often the sample off body tissue you have only contains a small amount of DNA. PCR is used to make many millions of copies quickly. PCR is an artificial way of DNA replication. PCR is a simple 3-stage process that is repeated times.

11 Stage 1 Denaturation. The DNA is heated to 95 o C and the strands separate as the H bonds are broken Stage 2 Attach primers. The solution is cooled to 50 o C and primers (nucleotide bases) bind to both strands of the DNA. Stage 3 Extension. Raise the temperature to 72 o C the new strands are completed.

12 Using Gel Electrophoresis it is possible to establish the order of each nucleotide in a strand of DNA (that is the sequence of the nucleotides). This can be used to map the location of genes as in a species based Genome project or to study a particular mutation or disorder. There are two techniques available: Manual Sequencing this is often called the Sanger method and uses gel electrophoresis. The gel is left to run for 1 3 hours. Small samples are sequenced by a trained technician analyzing the location of the different nucleotides.

13 Automatic Sequencing when a large amount of DNA needs to be sequenced this can be done reasonably quickly by machine. The four nucleotides are fluorescently labeled and a laser scanner reads the single lane of a gel sending the information to a computer which reconstructs the base sequence. These machines can be left to run for 10 hours at a time.

14 These techniques are now commonly used by Forensic Scientists to assist the police to gather evidence to solve crime. It must be stated that this is only more evidence in a collection of evidence that may indicate a particular person was present at the scene of a crime.

15 A gene probe is a small piece of single stranded DNA or RNA that has been radioactively labelled or labelled with a flourescent marker. Gene probes are used to locate target sequences of DNA. They tend to be nucleotides in length, but can be up to 1000 and are complementary for a known sequence. The sequence must be complementary to the target sequence to be able to find the correct piece of DNA in the sample being studied.

16 There are regions in DNA which vary in length in individuals, as they contain repeated sequences of DNA eg GAGAGAGA. These regions are inherited, just like alleles, thus individuals are unique. The size of the repeated sequences can be shown on an electrophoresis gel and this is known as a DNA Profile. There are two types:

17 STR short tandem repeats section of noncoding DNA of between 2 and 5 bases. For example, the dinucleotide GA is repeated many times to form the STR. These are inherited and an individual has a distinctive number based upon heredity. VNTR variable nucleotide tandem repeats also a section of non-coding DNA, but they are longer than 5 bases.

18 Cloning means to make an identical copy. By using these techniques it has become possible for scientists to remove sequences of nucleotides which are known to code for a particular protein, insert them in another organism and have the gene function to produce a particular effect An example of this, described above, is the addition of the human insulin gene to a bacterial plasmid so that the bacteria can be used to produce insulin for use by diabetics. To achieve this scientists needed to sequence the gene for human insulin, find restriction enzymes that would cut the DNA in the appropriate places, find a gene probe that could be used to remove the insulin gene and then transform the bacterial plasmid.

19 Vectors are commonly used to transport DNA from one organism to another There are two main types of vectors: plasmids and viruses. Plasmids: These are small circular parts of DNA that have come from bacteria. Genes can be inserted into these plasmids (using restriction enzymes and DNA ligase) and transported to another organism or put back into the bacteria, once it has been modified. Plasmids are commonly used by genetic engineers. For example, Insulin is now produced in vast quantities using bacteria. The human gene for the production of insulin is inserted into a plasmid. The bacteria are then grown with the insulin gene and caused to produce insulin which is harvested and sold to insulin diabetics. In the past insulin was taken from the pancreas of dead pigs, and sometimes sheep and cattle. It was expensive, some people had allergic reactions to it and others had religious, moral or ethical objections to using the animal insulin.

20 Viruses by nature infect other cells and insert their DNA or RNA in the process. Genetic engineers can insert foreign DNA into viruses and target cells. An attempt was made to use viruses as a vector to treat Cystic Fibrosis. Individuals with this disease produce a very thick mucous in their respiratory system (as well as thick mucous at other mucous membranes and other symptoms) due to a point mutation. The viruses were transformed to carry the correct gene coding for the protein that makes the mucous and inhaled by the sufferer. A number of problems occurred with this gene therapy and it was withdrawn from sale.

21 MEDICINAL Treatment of Cancer Pharmaceuticals Rational Drug Design Treatment of Diseases Stem Cell research Malnutrition is caused by both the lack of food and the lack of food with sufficient nutrition value. Many crop species, including rice, wheat and sorghum common food staples in many cultures, are being transformed to become richer sources of nutrients such as Vitamin A. AGRICULTURE Many agricultural crop species are being transformed to confer protection against diseases such as cotton, canola, corn and wheat. CONSERVATION For a species to be viable, it requires genetic diversity. Researchers are using the DNA profiles of many endangered species to better manage the existing populations.