Advanced Molecular Biological Techniques

1. The Polymerase Chain Reaction (PCR) Restriction Fragment Length Polymorphism Sequencing of DNA bases Transformation (By Emily Kennedy) Advanced Molecular Biological Techniques By Ruba Safieddine

2. The Polymerase Chain Reaction (PCR) • Developed by Mullis, who was awarded the Noble Prize in 1992 • PCR is a process that allows the production of many copies of DNA fragments • PCR uses heat (temperatures between 94 º C -96 º C) to separate the 2 strands of DNA by breaking the H-bonds between complimentary base pairs

3. The two strands are used for replication • Temperature is lowered to 50 º C- 60 º C and DNA primers attach to the DNA templates. • At 72 º C Taq polymerase identify the DNA primers and begin to attach free nucleotides to complete the complimentary strand of each template. • When the replication is complete, the process is repeated. Since each new cycle has more templates that can be used for replication, each cycle results in an “exponential increase in the number of copies of the target DNA”

4. This process can result in 2 types of strands: Variable-length strands: A mixture of strands of DNA that have been replicated and are of unequal length Constant length strands: A mixture of strands of DNA that have been replicated and are of equal length

5. Uses of PCR: • Forensic criminal investigations: a single cell can be replicated many times, therefore only a small sample of DNA evidence is required from a crime scene • Medical Diagnosis: to detect the presence of HIV • Genetic Research uses fossil remains to determine if 2 species are closely related Video of PCR: http://www.youtube.com/watch?v=_YgXcJ4n-kQ&feature=related

6. Restriction Fragment Length Polymorphism RFLP • Polymorphism: any difference in DNA sequence that can be detected between individuals (in the coding or non coding region)  can be used in coding regions to detect specific mutations in an individual e.g. Sickle cell anemia • Polymorphic: a term used to describe the genomes of individuals from the same species

7. RFLP analysis is the comparison of the differences in the length of DNA fragments between different people using the following techniques: • Step 1: DNA is extracted from sample and then digested by restriction enzymes called endonucleases • Step 2: The DNA is run on gel electrophoresis where it appears as a blob (since there are so many fragments and they are so close in size) • Step 3: Double stranded DNA is placed in a denaturing solution causing it to become single stranded. Using a process called southern blotting, an electric current allows the DNA in the gel to transfer to a nylon membrane. • Step 4: The nylon membrane is put in a solution with radioactive nucleotide probes that attach at specific locations. • Step 5: Hybridization occurs complimentary base pairing through the formation of hydrogen bonds between the probes and the DNA

8. Step 4: The nylon membrane is put in a solution with radioactive nucleotide probes that attach at specific locations. • Step 5: Hybridization occurs complimentary base pairing through the formation of hydrogen bonds between the probes and the DNA • Step 6: The nylon membrane is placed against x-ray film where an autoradiogram (gel pattern on the x-ray film) is formed * This pattern can be used in forensic investigations to connect a suspect to a crime scene based on a DNA match. It can also be used to find a mutation that may cause a genetic disorder. • Diagram of RFLP : http://homepage.smc.edu/HGP/images/rflp.gif

9. Sequencing of DNA Bases • determining the exact sequence of nucleotide bases in a specific DNA strand • Main method Dideoxy Sanger method developed by Fred Sanger in 1977

10. Dideoxy Sanger method: -Four copies of a single stranded DNA with a radioactively labeled primer on its end are placed in 4 reaction mixtures -Each test tube contains DNA polymerase, all four deoxynucleoside triphosphates (dNTP) e.g. Adenine (dATP) and one radioactively labeled dideoxy analogue (either dideoxy-adenine (ddATP) , dideoxy-thymine (ddTTP), dideoxy- guanine (ddGTP), or dideoxy-cytosine (ddCTP). -A dideoxy analogue has a ribose sugar that lacks an –OH group on the 2’ and 3’ carbon Therefore synthesis of a new DNA strand stops because the next complementary base cannot be added by the DNA polymerase

11. -Since only a small portion of the mixtures contain dideoxy analogues, different length of complimentary DNA will form depending on when the ddNTP is integrated. e.x. 3’ G G A C T A T C C A T T A G C 5’ Strands that may be produced: 5’ C C T G C C T G A T A G C C T G A T A G G C C T G A T A G G T A A T C G 3’ Strand to be replicated is placed in test tube with dideoxy analogue Guanine  The second the dideoxy analogue is integrated the chain is prevented from growing any further

12. -Next, gel electrophoresis is used to separate the strands of different length • Once this is done they are placed against an X-ray film where the pattern can be determined by method of autoradiogram Video showing process: http://www.youtube.com/watch?v=oYpllbI0qF8

13. Transformations • Transformation is the process of bringing in foreign DNA from another source. • This commonly occurs in bacterial cells when a plasmid or a virus is inserted which changes or introduces a new characteristic to the bacteria cell. • The plasmid or the virus is known as a vector, which carries the DNA into the host cell. • Once the bacterium takes this foreign DNA it is called a competent cell. But not all bacteria will take this new DNA, so transformation does depends on the cell being competent.

14. Although, cells can be encouraged to take this DNA and it is through working with the charges of the ions and working with the ranges of temperatures. This is done by:-Having DNA and cell membranes with both slightly negative charges -When the plasmid with the DNA and the bacteria is put in CaCl2 solution, the positive calcium ions will attract to the negative charged plasmid which is give a neutral charge. -By having the solution at a low temperature and then dramatically and rapidly increasing it, a draft is created that allows the plasmid to move through the pores of the cell membrane and into the cytoplasm.

15. To see if transformation did successfully occur you can perform a test that is illustrated at this website: http://www.biotechlearn.org.nz/var/biotechlearn/storage/images/themes/from_genes_to_genomes/images/bacterial_transformation/4063-1-eng AU/bacterial_transformation_large.jpg Basically, this test uses the principle of comparing your samples. • First have a Petri dish of the bacteria that does not have the plasmid containing the genetic information. • Then compare it to a Petri dish of your sample of bacteria that you encouraged transformation. • Depending on what the plasmid or virus’s DNA information was, it will be different from your original bacteria.

16. Let’s say the plasmid contains an antibiotic in the genetic code of the DNA, your second Petri dish should have a noticeable decrease in surface area of the bacteria. • If there is a decrease, transformation was successful. *Of course, not all of the bacteria should have disappeared because not every bacteria cell was transformed. • More recently, electric shock has encouraged transformation in plant cells through electroporators. By sending electric shocks, the strong cell walls loosen and it becomes easier for the foreign DNA to get through.

17. They can also do this by using a gene gun to break the ridge walls. They do this by: -Taking a water droplet and charging it. -Send it through a tube with an electrical discharge. -At the end of the tube is a sheet that has DNA wrapped in gold particles. They use gold because it is very non-reactive. -Then the water droplet that contains these DNA wrapped gold particles is propelled at the cells and the force of the impacts breaks the cell walls and cell membranes.

18. References Di Giuseppe, Maurice, Vavitsas, A., Ritter, Dr. Bob, & F., Douglas (2003). Biology 12. Canada: Thomson Nelson. * All definitions are directly from the text*