RT-PCR lab You have a cell…is a certain gene on (by “on,” we mean active and producing mRNA?)? If a certain gene is on when the cell divides, the gene.
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Presentation on theme: "RT-PCR lab You have a cell…is a certain gene on (by “on,” we mean active and producing mRNA?)? If a certain gene is on when the cell divides, the gene."— Presentation transcript:
RT-PCR lab You have a cell…is a certain gene on (by “on,” we mean active and producing mRNA?)? If a certain gene is on when the cell divides, the gene might produce a protein that causes cell division….
Central Dogma: DNA has genes and is in nucleus TRANSCRIPTION: Double strands of DNA unwind to allow synthesis of messenger RNA (mRNA) from one strand (the coding strand) The mRNA moves out of the nucleus to the cytoplasm mRNA binds to Ribosomes to code for a protein- protein made (translation) Protein carries out intent of gene (red hair protein = hair gene)
Transcription: Unwind 2 DNA strands and copy one making mRNA (ch.18)
So, first step of RT PCR is: ISOLATE THE mRNA from the cell Next, make DNA from the mRNA This is reversing “transcription”– so use an enzyme originally obtained from viruses– ENZYME IS CALLED REVERSE TRANSCRIPTASE (THE RT OF RT PCR)
Last slide: this is the RT part of RT PCR PCR part: After RT, you now have a tiny, trace amount of what is called complimentary DNA (cDNA). This tiny trace amount is not enough to sequence. Next, you have to make enough copies of the tiny trace amount of cDNA to sequence
Steps in PCR (fig. 19A01): Target sequence By using Specific primers To the target Sequence
Now repeat cycle over and over Get huge number of DNA copies --enough that you can now study The gene by sequencing it (finding Order of nucleotides)
PCR: polymerase chain reaction- making many copies of cDNA View animation of PCR: best: http://www.dnalc.org/ddnalc/resources/shockwave/pcran whole.htmlhttp://www.dnalc.org/ddnalc/resources/shockwave/pcran whole.html OK: http://users.ugent.be/~avierstr/principles/pcrani.html http://www.people.virginia.edu/%7erjh9u/pcranim.html http://www.abpischools.org.uk/resources/poster- series/pcr/pcranim.asphttp://www.abpischools.org.uk/resources/poster- series/pcr/pcranim.asp PCR animation links http://www.dna.utah.edu/PCR_Animation_Links.htm
Summary of RT PCR RT-PCR animation http://www.bio.davidson.edu/Courses/gen omics/RTPCR/RT_PCR.htmlhttp://www.bio.davidson.edu/Courses/gen omics/RTPCR/RT_PCR.html
Electrophoresis to separate DNA by size (remember our prior discussion and animation): Electrophoresis to separate DNA by size (remember our prior discussion and animation): The fragment we want should be Of a known size!!