SCIE1106 Lecture Notes - Lecture 18: Thermus Aquaticus, Complementary Dna, Ribonuclease H
L18 Copying DNA & RNA
in vitro
•
Know components of PCR
•
Know steps of PCR
•
Draw, illustrate binding of PCR primer
pairing to a complementary ds template and
extension of the primers, by Taq polymerase
•
Know properties of RT
•
Know differences between PCR/ RT-PCR, and
when each is used
PCR
Requirements
1. Primers
a. Need sequence in formation to
initiate/design
b. 20-30 nt
c. bind complementary sequences on
template ssDNA
d. primers- short strands of RNA
2. DNA polymerase
a. Heat stable
b. From thermophilic Thermus
aquaticus (Taq) (able to withstand
and function under high
temperature) makes mistakes in
1/400 nt
3. dNTPs precursors /building blocks
4. thermocycler / aq environment
5. buffer
6. DNA template
Incoming nt comes to 3’ end, and moves from 5’ to 3’
Runs for 20-40 cycles, having 220 to 240 copies of
original template
Functions
1. To clone a genomic DNA fragments for DNA
library (cDNA)
2. Forensic science
3. Environmental studies
a. PCR can be used, as different
molecular markers for different
microbes/fungi
b. Nutrient cyclilng interrestrial &
aquatic ecosystems
c. Environmental impacts
d. Rehabilitation
e.g with different lengths of PCR products show
greater diversity (healthier soil) and with PCR
products having similar sizes, will show little
diversity (contaminated soil)
Reverse Transcriptase RT (know the properties)
• Reflect the genes, expressed by tissues/cells
in a sample (gives an idea of gene expression
because we are using RNA for PCR)
• cDNA= complementary DNA
• are RNA-dependent, DNA polymerases
o RT is the enzyme, that depends on
RNA, to make copies of DNA
• found in retroviruses
• requires primer
• note: RNA cant be used in PCR – r.E doesn’t
dissolve RNA, gotta convert RNA to cDNA to
use it in PCR ( we use RT)
1. add first primer that is poly T to bind to poly
A (to make DNA)
2. Buffer
3. PCR tube
4. Enzymes (RT, DNA polymerase)
5. dNTP
DNA now makes a new strand at 3’, from 5’ to 3’
Get cells from tissues
Get mRNA (with poly A tail)
Hybridize with poly T → now act as a primer
(primer is short dsRNA/ dsDNA), to initiate binding
of RT in this case (DNA polymerase later on)
DNA copy is made using RT (aka cDNA) (hybrid of
mRNA and cDNA)
RNA is degraded with RNase H
RNA is fragmented and have leftover, blue strands
that act as primers for DNA polymerase (*not RT*)
to add nt bases to the second strand
So then there is less mRNA on a ds strand
Now the product is called cDNA copy of original
mRNA
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