Polymerase Chain Reaction 1

Being able to clone target sequences gives scientists the opportunity to do a lot of analytical things, such as:

- Identification of genetic defects and viruses in human cells
- COVID-19 testing
- MTB Diagnosis
- Used in evolutionary studies and in forensics

- Gold standard tests when testing for MTB complexes.
- A purely automated technique.

Xpert MTB / RIF Assay (Radio Immunofluorescent Assay)

Xpert MTB / RIF Assay tests resistance to ___, one of the most effective drugs to treat tuberculosis.

rifampin (rifampicin)

For a medical technologist to be able to use Xpert MTB / RIF Assay, one has to be trained by ___.

RITM (Research Institute for Tropical Medicine)

Basic Principle of PCR:

It begins with just one template and from that one template is the first cycle with two copies. The second cycle is going to be four copies. Third cycle, it's going to multiply continuously

PCR usually takes ___ cycles

20 to 30

REACTION COMPONENTS

A. THERMAL CYCLER
B. DNA TEMPLATE
C. DEOXYNUCLEOTIDE TRIPHOSPHATES (dNTPS)
D. DNA POLYMERASE
E. PRIMER
F. PCR BUFFER

Thermal cyclers are laboratory instruments that facilitate the PCR processes of:

denaturation, annealing, and elongation

Thermal cyclers are also known as

thermocyclers or PCR machines

Thermal cyclers are called thermal because the ___ throughout the entire PCR process

temperature always changes

Contains the target DNA Region that you want to amplify.

DNA TEMPLATE

Deoxynucleotide triphosphates as building blocks of the new DNA strands:

dATP, dCTP, dGTP, and dTTP

Deoxynucleotide triphosphates are added to your PCR reaction in ___ amounts.

equimolar

Does not have the hydroxyl component.

Dideoxynucleotide (ddNTP)

Contains a hydroxyl group.

Deoxynucleotide (dNTP)

Not used for DNA replication in Polymerase Chain Reaction, since the goal here is to amplify. However, this is used in in vivo DNA replication.

Dideoxynucleotide (ddNTP)

Used for DNA replication in Polymerase Chain Reaction.

Deoxynucleotide (dNTP)

DNA replication occurs in the ___, near connective tissues with nutrients. ddNTPs can help replenish dead cells, but uncontrolled replication could cause ___.

stratum basale
basal cell carcinoma

DNA Polymerase adds nucleotides to the ___ of the complementary DNA strand

3' end

Compared to DNA replication, PCR is ___.

specific

The first paper was published for using TAQ DNA Polymerase for the PCR.

1988

1989 Molecule of the Year

DNA Polymerase

OTHER POLYMERASES USED

1. TAQ DNA Pol (Thermus aquaticus)
2. Pfu DNA Pol (Pyrococcus furiosus)
3.

Despite the thermostability of TAQ DNA polymerase, it is still subject to loss because enzymes are ___

very sensitive samples.

Thermostable and has an exonuclease activity.

Pfu DNA Pol
Pyrococcus furiosus

DNA polymerase runs from ___, but due to the exonuclease activity of Pfu DNA, it runs from ___

5' to 3'
3' to 5'

The larger fragment from Escherichia coli

DNA Pol1 Klenow Fragment

Primers attach to the nucleotide bases through the ___.

polymerases

Primers add to the specificity of the PCR run since it directs which region to ___.
Primers dictate the polymerase to tell when to ___.
Primers are ___ to the target DNA sequence.

amplify
stop
complementary

Primers can be designed ___ or ___

manually
generated through a software program

There is an available database containing the primers for different organisms.
This is where we get the sequences for primers.

National Center for Biotechnology Information (NCBI)

Primers can be designed ___ or generated through a software program

manually

Primers can be designed ___ or ___

manually
generated through a software program

There is an available database containing the primers for different organisms.
This is where we get the sequences for primers.

National Center for Biotechnology Information (NCBI)

2 KINDS OF PRIMER

Forward Primer
Reverse primer

It provides optimal conditions.

PCR BUFFER

ENZYMES PRESENT IN DNA REPLICATION BUT NOT IN PCR

DNA Ligase
Helicase
Topoisomerase

An in vitro technique which involves the amplification of a specific DNA region or sequence through a series of temperature cycles.

POLYMERASE CHAIN REACTION

The counterpart of Polymerase Chain Reaction is ___, which occurs inside the body.

DNA Replication

bacteria that is responsible for tuberculosis.

Mycobacterium tuberculosis

PCR COMPONENTS

- DNA Sample
- Primers
- Nucleotides
- Taq Polymerase
- Mix Buffer
- PCR Tube
- Thermal Cycler

In DNA Replication, these are used as the building blocks.

dNTPS
deoxynucleotides

An enzyme which synthesizes a long chain of nucleic acids and catalyzes the elongation of DNA.

DNA POLYMERASE

Hybridizes to the complementary strand or to the 5' end.
Binds to the Antisense strand.

Forward Primer

Hybridizes to the DNA target region at the 3' end.
So it binds to the Sense strand.

Reverse primer

He isolated TAQ DNA Polymerase from the bacteria Thermus aquaticus

Thomas Brock (1969)

Thomas Brock (1969) isolated this bacteria in a geothermal hot spring found in Yellowstone National Park in Montana, USA.

Thermus aquaticus

Won a Nobel Prize in chemistry, for inventing the PCR technique in 1993 while working for CETUS Corporation.

Kary Mullis (1983)

attach to the target region, speeding up the reaction when two oligonucleotides are produced.

short single-stranded DNA (oligos) or primers

After primers attach, ___ extends the DNA, adding nucleotide bases on both strands, similar to normal replication

DNA polymerase

___, the building blocks of DNA, are added for this process.

dNTPs (deoxynucleotide triphosphates)

Mullis designed primers to study ___

point mutations

A type of polymerase from the bacteria Escherichia coli.

Klenow Fragment

Before the use of Taq polymerase, the Klenow fragment was used in the PCR process. However, this is ___, and not ideal when the PCR process needs to undergo different temperature cycles.

not heat stable

First commercial thermal cycler developed by Perkin Elmer Cetus

TC1 DNA Thermal Cycler

When was the first commercial thermal cycler developed?

1987

Randall. Saiki and K. Mullis paper title

"Primer-directed enzymatic amplification of DNA with a thermostable DNA Polymerase"

isolated the pure and native Taq DNA Polymerase from Thermus aquaticus.

Dr. Henry Erlich, Dr. Gelfand, Dr. Susan Stoffel

In vitro =

outside the body

PCR is the generation of millions of copies of your target ___.

DNA regions