Transcript RT-PCR lab - University of Colorado Denver

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)
DNA Structure
DNA is two
strands of
nucleotides
Wrapped around
each other in a
double helix
We sequence the
DNA to find out
about the genes
present
(later:
bioinformatics lab)
Unwind, mRNA is
made off DNA
template- similar to
this picture of DNA
made off of DNA.
Nucleotides pair up:
G always pairs with
C, T pairs with A.
Except in RNA, T is
replaced with U.
Transcription:
RNA synthesis
(note coding
and template
strands)
(ch.21)
Making mRNA off DNA:
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 reactionmaking many copies of cDNA
• View animation of PCR:
• best:
• http://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/posterseries/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/geno
mics/RTPCR/RT_PCR.html
Electrophoresis to separate DNA
by size (remember our prior discussion and animation): The
fragment we want should be Of a known size!!