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!!