lecture 5 - 08/10/24 Flashcards
What is in vitro?
in the lab
What is in vivo?
in organism
ingredients for PCR
Template DNA
Primers
dNTPs
Buffer (Mg 2+)
taq polymerase
What is taq polymerase?
modified DNA polymerase
from bacterial species that can survive hotter temperatures (originally in hot springs, Yellowstone)
What are the 3 steps in PCR?
- Denaturation
- Annealing
- Extension
Denaturation
double DNA strand melts open
Annealing
primers bind to DNA and polymerase attaches and starts copying DNA
Extension
at 72oC: optimum temperature for taq polymerase and extension of fragment
What is the optimum temperature of DNA polymerase?
37oC (body temp)
Is PCR amplification linear or exponential?
exponential
e.g. 30 cycles = 20^30
Which bits of the DNA is amplified?
only DNA between primers is amplified
ends of the amplified fragment are defined by 2 primers
(as only replicating a specific section, NOT replicating the genome)
Primers in PCR
self designed to cater for the specific region needed
~20 bp oligonucleotides
What is agarose?
complex polysaccharide
separation of DNA by agarose gel electrophoresis
electric current is applied to gel
DNA moves to + electrode because it is - charged
moves through gel depending on:
- conformation (linear, circular, supercoiled)
- size
How does size affect DNA movement in agarose gel electrophoresis?
smaller fragments move through the gel faster than large ones
How to detect DNA in agarose gel electrophoresis?
stain DNA with fluorescent dye for detection by UV exposure
e.g. ethidium bromide - intercalates + binds w/ DNA
What is the ladder used for in agarose gel electrophoresis?
used to identify each band size
- standardises sizes
Uses of PCR
detection of pathogens in water
DNA sequencing
Diagnosis of genetic disorders
Prenatal diagnosis
Analysis of ancient DNA
Genetic fingerprinting
Forensic analysis
Limitations of PCR
- sequence information is required to design 2
primers - limit on length of amplified fragment
- potentially high error rate e.g. Taq polymerase =
~10^-4 - very sensitive to exact reaction conditions, not
easily quantified - tiny amounts of contaminating DNA will also be
amplified
Sanger Sequencing ‘ingredients’
DNA polymerase
dNTPs +ddATP, ddTTP, ddGTP, ddCTP
template DNA
Primer
DNA synthesis 5’ to 3’ direction
What is a dNTP?
normal deoxyribonucleoside triphosphate
3’ OH allows strand extension at 3’ end
What is a chain-terminating ddNTP?
chain-terminating dideoxyribonucleoside triphosphate
coloured
terminates the sequence as no 3’ -OH
The principle of Sanger sequencing
- add primer to single-stranded DNA fragment to
be sequenced - add small amounts of labelled chain-terminating
ddNTPs - add excess amounts of unlabelled dNTPs
- mixture of DNA products (each containing a
chain-terminating ddNTP labelled with a specific
fluorescent marker - products loaded onto capillary gel
- electrophoresis, size-separated products are
read in sequence
What shape chromosome are bacterial genomes?
circular
