RT-PCR lab
description
Transcript of RT-PCR lab
RT-PCR labRT-PCR labYou have a cell…is a certain gene You have a cell…is a certain gene on (by “on,” we mean active and on (by “on,” we mean active and
producing mRNA?)?producing mRNA?)?If a certain gene is on when the If a certain gene is on when the
cell divides, the gene might cell divides, the gene might produce a protein that causes cell produce a protein that causes cell
division….division….
Central Dogma: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)
DNA StructureDNA Structure
DNA is two strands of nucleotidesWrapped around each other in a double helix
We sequence the DNA to find out about the genes present(later: bioinformatics lab)
Unwind, mRNA is Unwind, mRNA is made off DNA made off DNA
template- similar to template- similar to this picture of DNA this picture of DNA made off of DNA.made off of DNA.
Nucleotides pair up:Nucleotides pair up:G always pairs with G always pairs with C, T pairs with A. C, T pairs with A.
Except in RNA, T is Except in RNA, T is replaced with U.replaced with U.
Transcription:Transcription:RNA synthesisRNA synthesis
(note coding (note coding and template and template
strands)strands)(ch.21)(ch.21)
Making mRNA off DNA:Making mRNA off DNA:
Transcription: Unwind 2 DNA strands Transcription: Unwind 2 DNA strands and copy one making mRNA (ch.18)and copy one making mRNA (ch.18)
So, first step of RT PCR is: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):Steps in PCR (fig. 19A01):
Target sequenceBy using Specific primersTo the targetSequence
Now repeat cycle over and overGet huge number of DNA copies--enough that you can now study The gene by sequencing it (findingOrder of nucleotides)
PCR: polymerase chain reaction- PCR: polymerase chain reaction- making many copies of cDNAmaking many copies of cDNA
• View animation of PCR:• best:• http://www.dnalc.org/ddnalc/resources/shockwave/
pcranwhole.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.asp• PCR animation links• http://www.dna.utah.edu/PCR_Animation_Links.htm
Summary of RT PCRSummary of RT PCR
• RT-PCR animation• http://www.bio.davidson.edu/Courses/
genomics/RTPCR/RT_PCR.html
Electrophoresis to separate DNA Electrophoresis to separate DNA by size by size (remember our prior discussion and animation): (remember our prior discussion and animation): The
fragment we want should be Of a known size!!