Tutor task 2: Polymerase Chain Reaction and SNP
analyse
Assignment 1: PCR and multiplex PCR
Multiplex PCR:
Multiplex polymerase chain reaction (Multiplex PCR) refers to the use of polymerase chain reaction
to amplify several different DNA sequences simultaneously (as if performing many separate PCR
reactions all together in one reaction). This process amplifies DNA in samples using multiple primers
and a temperature-mediated DNA polymerase in a thermal cycler. The primer design for all primers
pairs has to be optimized so that all primer pairs can work at the same annealing temperature during
PCR. This technique requires two or more probes that can be distinguished from each other and
detected simultaneously. There is a range of different probe technologies available, all using
fluorophores.
Types of multiplex PCR:
1. Single template PCR reaction:
- This technique uses a single template which can be a genomic DNA.
- Along with several pairs of forward and reverse primes to amplify specific regions within a
template; in the same reaction tube.
2. Multiple template PCR reaction:
- This technique uses multiple templates.
- Several primers sets of forward and reverse primers for each template and regions within
the template; in the same reaction tube.
Probes:
Probes are primers with an addition on one group that allows us to see the primer. This change can
be a molecule that fluoresces and shines when hit with light, or it can be an attachment to a colored
bead, for example. This then allows us to visualize when a probe (primer with a colored bead
attached to it) attaches to DNA. Since we designed the probe, we can tell what the DNA sequence is
at that particular point in the DNA.
Questions:
How has the PCR protocol been adapted in comparison with the standard PCR protocol?
In multiplex PCR multiple primers are released on one or more pieces of DNA. As a result,
multiple pieces of DNA are amplified in 1 sample. If the sample is on gel, multiple straps will
arise.
analyse
Assignment 1: PCR and multiplex PCR
Multiplex PCR:
Multiplex polymerase chain reaction (Multiplex PCR) refers to the use of polymerase chain reaction
to amplify several different DNA sequences simultaneously (as if performing many separate PCR
reactions all together in one reaction). This process amplifies DNA in samples using multiple primers
and a temperature-mediated DNA polymerase in a thermal cycler. The primer design for all primers
pairs has to be optimized so that all primer pairs can work at the same annealing temperature during
PCR. This technique requires two or more probes that can be distinguished from each other and
detected simultaneously. There is a range of different probe technologies available, all using
fluorophores.
Types of multiplex PCR:
1. Single template PCR reaction:
- This technique uses a single template which can be a genomic DNA.
- Along with several pairs of forward and reverse primes to amplify specific regions within a
template; in the same reaction tube.
2. Multiple template PCR reaction:
- This technique uses multiple templates.
- Several primers sets of forward and reverse primers for each template and regions within
the template; in the same reaction tube.
Probes:
Probes are primers with an addition on one group that allows us to see the primer. This change can
be a molecule that fluoresces and shines when hit with light, or it can be an attachment to a colored
bead, for example. This then allows us to visualize when a probe (primer with a colored bead
attached to it) attaches to DNA. Since we designed the probe, we can tell what the DNA sequence is
at that particular point in the DNA.
Questions:
How has the PCR protocol been adapted in comparison with the standard PCR protocol?
In multiplex PCR multiple primers are released on one or more pieces of DNA. As a result,
multiple pieces of DNA are amplified in 1 sample. If the sample is on gel, multiple straps will
arise.
