RT-PCR lab

1
RT-PCR lab

• You have a cellis 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.

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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)

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DNA Structure
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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)
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Unwind, mRNA is made off DNA template- similar to
this picture of DNA made off of DNA.Nucleotides
pair upG always pairs with C, T pairs with A.
Except in RNA, T is replaced with U.
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TranscriptionRNA synthesis(note coding and
template strands)(ch.21)
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Making mRNA off DNA
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Transcription Unwind 2 DNA strands and copy one
making mRNA (ch.18)
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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)

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• 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

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Steps in PCR (fig. 19A01)
Target sequence By using Specific primers To the
target Sequence
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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)
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PCR polymerase chain reaction- making many
copies of cDNA

• View animation of PCR
• best
• http//www.dnalc.org/ddnalc/resources/shockwave/pc
  ranwhole.html
• OK
• http//users.ugent.be/avierstr/principles/pcrani.
  html
• http//www.people.virginia.edu/7erjh9u/pcranim.ht
  ml
• http//www.abpischools.org.uk/resources/poster-ser
  ies/pcr/pcranim.asp
• PCR animation links
• http//www.dna.utah.edu/PCR_Animation_Links.htm

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Summary of RT PCR

• RT-PCR animation
• http//www.bio.davidson.edu/Courses/genomics/RTPCR
  /RT_PCR.html

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Electrophoresis to separate DNA by size (remember
our prior discussion and animation) The fragment
we want should be Of a known size!!