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Transcript
GENERATING OF MELANOMA MOUSE MODELS EUNICE ASARE Dr Alan Houghton Laboratory Memorial Sloan Kettering Cancer Center (MSKCC) ABSTRACT Our ongoing project is to develop a line of transgenic mice with MutHbraf to express melanoma that mimic the same situation in human patients. A line of transgenic mice have been developed and we are genotyping them to check for the presence of the MutHbraf gene using Polymerase Chain Reaction (PCR) method. We will cross breed MutHbraf transgenic mice with mice whose Ink4a gene ( (tumor suppressor gene) has been “Knocked Out”. New mice from the cross will be monitored to see if they develop melanoma. The melanomas developed will be characterized and treated with the HGP75 optimized or HGP75 vaccine to suppress the melanoma. DETECTION AND ANALYSIS OF PCR PRODUCT Gel Electrophoresis :It is a method to separate DNA strands by size,and to determine the size of the separated strands by comparison to strands of known length. PCR CYCLE The commercial blue juice dye used to load PCR products into agarose gel, often overlaid our expected DNA bands, so it made it difficult to genotype our mice. Instead, my mentor suggested we use GLYCEROL with XYLENE CYANOL to load our PCR samples. Not only is it inexpensive to prepare but also the Xylene dye does not overlay the DNA bands and can be clearly visualized. USES OF PCR PRODUCT Genetic Finger printing Paternity testing Detection of hereditary disease Study the functions of genes Genotyping transgenic mice : An example is the Ink4a(tumor suppressor gene) PCR I performed to check the genotype of KO mice. The mice KI-613 and KI-436 had positive bands for both wt and neo; they are heterozygous for INK4a gene. KI-433 did not have a band for the wt but had a band for the neo; it’s a true knockout. The primary goal of the PCR technique is to amplify a known/target sequence of DNA. AFTER 25 – 30 CYCLES THE TARGET DNA WILL BE EXPONENTIALLY AMPLIFIED. THE NUMBER OF COPIES PRODUCED WILL BE BILLIONS Eg. 230 = 1073,741,824 COMPONENTS FOR A PCR DNA TEMPLATE: A well defined part of a DNA fragment containing INK4a WT 273 bp the sequence which will be amplified. Magnesium Chloride(Mgcl2) PRIMERS: DNA strands--not more than fifty (usually 18-25 bp) Stabilizes the primer template bond formed and enables the DNA polymerase to work at its optimum. nucleotides that exactly match the beginning and end of the DNA fragment to be amplified. To have a good yield of PCR product considerations for constructing a primer include: 10x Buffer: Provides a •GC-content should be between 40-60. •Calculated Tm (temperature at which 50% of the primer molecules are annealed to the DNA template) for both primers used in reaction should not differ >5C and Tm of the amplification product should not differ from primers by >10C. To calculate the Tm for both primers the formula used depends on the type of PCR: Tm =2(A+T) +4(G+C) OR Tm= 81.5+ 0.41(%GC)675/N *N is the primer length in bases. •Annealing temperature usually is -5C the calculated lower Tm. However it should be chosen carefully for individual conditions. Inner self-complementary hairpins of >4 and of dimers >8 should be avoided. •3' terminus is extremely case sensitive - it must not be complementary to any region of the other primer used in the reaction and must provide correct base matching to template. •3’-end Sequence- 3’ position in PCR primers is essential for the control of mis-priming (5').The inclusion of a G or C base at the 3' end of primers: This G/C helps to ensure correct binding at the 3' end due to the stranger hydrogen bonding of G/C. It also helps to improve the efficiency of the reaction by minimizing any error that might occur. Deoxynucleotide Phosphate (dNTP) : Nucleotide base pair (comparable to the building block of DNA) that form the new complementary strand for DNA template.The base pairs Adenine has its complementary strand of Thymine in DNA, but in RNA Thymine is replaced by Uracil, and the complementary strand of Guanine is Cytosine. The base pairs adhere to the DNA template during extension in the PCR cycle DNA Polymerase : Enzyme obtained from Thermus Aquaticus called Taq. It picks up the nucleotide base pairs and “screws” it to the 3’ end of the DNA template to form a complementary strand. INK4a neo 300 bp : FUTURE DIRECTIONS Since we used the Polymerase Chain Reaction (PCR) method to also detect the presence of MutHbraf in our obtained transgenic mice we will now : MutHbraf transgenic mice with mice whose Ink4a gene ( tumor suppressor gene) has been “Knocked Out”. suitable chemical environment by maintaining the pH of the reaction in the ranges of 6-8pH. • We will cross breed MutHbraf transgenic mice with mice whose Ink4a gene ( tumor suppressor gene) has been “Knocked Out”. Nuclease free H2O: • New mice breed from the cross will be monitored to see if they develop melanoma. Serves as the primary solvent for the reaction, regular water will degrade the DNA template. • The melanomas developed will be characterized and treated with the HGP75 optimized or HGP75 vaccine to suppress the melanoma. PCR Machine: REFERENCES AKNOWLEDGEMENT Regulates the temperature of the reaction ELVA BARREDA and TAHA MERGHOUB MINYI TAN BARTEK JABLONSKI ALAN HOUGHTON LABORATORY OF IMMUNOLOGY MSKCC Dr. SAT BHATTACHARY HARLEM CHILDREN SOCIETY http://en.wikipedia.org/wiki/Polymerase_chain_reactionhttp://oregonstat e.edu/instruct/bb494/pcr.html http://www.accessexcellence.org/LC/SS/PS/PCR/PCR_technology.html www.invitrogen.com/pcr http://www.eppendorfna.com/applications/pcr_appl_primer.asp?SX=NF& Dieffenbach, C.W., Lowe, T.M.J., Dveksler, G.S., General Concepts for PCR Primer Design, in PCR Primer, A Laboratory Manual, Dieffenbach, C.W, and Dveksler, G.S., Ed., Cold Spring Harbor Laboratory Press, New York, 1995, 133-155. Innis, M.A., and Gelfand, D.H., Optimization of PCRs, in PCR protocols, A Guide to Methods and Applications, Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J., Ed., CRC Press, London, 1994, 5-11. Sharrocks, A.D., The design of primers for PCR, in PCR Technology, Current Innovations, Griffin, H.G., and Griffin, A.M, Ed., CRC Press, London, 1994, 5-11. Suggs, S.V., Hirose, T., Miyake, E.H., Kawashima, M.J., Johnson, K.I., and Wallace, R.B., Using Purified Genes, in ICN-UCLA Symp. Developmental Biology, Vol. 23, Brown, D.D. Ed., Academic Press, New York, 1981, 683. Kwok, S., Kellog, D.E. McKinney, N., Spasic, D., Goda, L., Levenson, C., and Sninsky, J.J., Effects of primer-template mismatches on the polymerase chain reaction: Human Immunodeficiency Virus 1 model studies. Nucleic Acids Res. 18:999-1005, 1990. http://www.life.uiuc.edu/molbio/geldigest/electro.html http://www.promega.com/paguide/chap1a4.pdf http://www.idtdna.com/Catalog/NucleaseFreeBuffers/Page1.aspx http://www.bio.calpoly.edu/ubl/Alu.htm