PCR
(Polymerase Chain Reaction)
Introduction to PCR Lysis buffer K used for blood cells and
PCR is a technique used to amplify (make viruses contains PCR buffer (50mM KCl,
multiple copies) of a specific DNA fragment in 10-20mM Tris-Cl, and 2.5mM MgCl2);
vitro 0.5% Tween-20 and 100 µg/mL
The technique was invented by Kary Mullis in the Proteinase K
1980s b Plasmid DNA and Chromosomal DNA
The principle of PCR is based on repeated Isolation method: The process of
thermal cycles breaking down cell walls followed by
The PCR machine is called a Thermocycler or separation of chromosomal DNA or
Mini Cycler plasmid DNA from other components
2 Primers
Primers are short DNA sequences that serve
as starting points for the synthesis of new
DNA copies
e rs PCR primers must be: 1) Specific, 2) Length
cl
o cy between 18-30 bases, 3) Tm difference of
m about 5°C between forward and reverse
er
Th primers, 4) GC content, 5) Secondary
structure and 6), Bonding at the 3' end
Components of PCR
3 dNTPs (deoxynucleotide triphosphates)
Function: The main building blocks for DNA
or RNA synthesis
dNTPs consist of:
MgCl₂ dATP (deoxyadenosine triphosphate)
DNA Polymerase dTTP (deoxythymidine triphosphate)
Enzyme Magnesium chloride
dCTP (deoxycytidine triphosphate)
dGTP (deoxyguanosine triphosphate)
G C T A
Buffer dNTPs
4 MgCl2 (Magnesium chloride)
Function: Magnesium ions (Mg2+) act as
cofactors that stimulate DNA polymerase
DNA Template Primers activity
Tube
Magnesium ions help strengthen the bond
between primers and form complexes that
1 DNA Template facilitate the incorporation of dNTPs into the
Function: Serves as a template for the DNA strand
formation of new, identical DNA molecules
Templates that can be used include DNA 5 PCR Buffer
fragments, plasmid DNA, or chromosomal Function: Maintains a stable pH environment
DNA suitable for enzyme function
Methods for preparing DNA templates Generally, PCR buffers already contain
a DNA Fragment MgCl2, but it is better if MgCl2 and PCR
Lysis method: A process of breaking buffer are separated so that variations in
down cell walls to extract DNA without concentration can be easily made
damaging it
1 Bioin
(Polymerase Chain Reaction)
Introduction to PCR Lysis buffer K used for blood cells and
PCR is a technique used to amplify (make viruses contains PCR buffer (50mM KCl,
multiple copies) of a specific DNA fragment in 10-20mM Tris-Cl, and 2.5mM MgCl2);
vitro 0.5% Tween-20 and 100 µg/mL
The technique was invented by Kary Mullis in the Proteinase K
1980s b Plasmid DNA and Chromosomal DNA
The principle of PCR is based on repeated Isolation method: The process of
thermal cycles breaking down cell walls followed by
The PCR machine is called a Thermocycler or separation of chromosomal DNA or
Mini Cycler plasmid DNA from other components
2 Primers
Primers are short DNA sequences that serve
as starting points for the synthesis of new
DNA copies
e rs PCR primers must be: 1) Specific, 2) Length
cl
o cy between 18-30 bases, 3) Tm difference of
m about 5°C between forward and reverse
er
Th primers, 4) GC content, 5) Secondary
structure and 6), Bonding at the 3' end
Components of PCR
3 dNTPs (deoxynucleotide triphosphates)
Function: The main building blocks for DNA
or RNA synthesis
dNTPs consist of:
MgCl₂ dATP (deoxyadenosine triphosphate)
DNA Polymerase dTTP (deoxythymidine triphosphate)
Enzyme Magnesium chloride
dCTP (deoxycytidine triphosphate)
dGTP (deoxyguanosine triphosphate)
G C T A
Buffer dNTPs
4 MgCl2 (Magnesium chloride)
Function: Magnesium ions (Mg2+) act as
cofactors that stimulate DNA polymerase
DNA Template Primers activity
Tube
Magnesium ions help strengthen the bond
between primers and form complexes that
1 DNA Template facilitate the incorporation of dNTPs into the
Function: Serves as a template for the DNA strand
formation of new, identical DNA molecules
Templates that can be used include DNA 5 PCR Buffer
fragments, plasmid DNA, or chromosomal Function: Maintains a stable pH environment
DNA suitable for enzyme function
Methods for preparing DNA templates Generally, PCR buffers already contain
a DNA Fragment MgCl2, but it is better if MgCl2 and PCR
Lysis method: A process of breaking buffer are separated so that variations in
down cell walls to extract DNA without concentration can be easily made
damaging it
1 Bioin
