3/24/2011

Detection Based on PCR

Technique

Ken Ratih Probosari

Novita Dewi

kenratih@its-indonesia.com
novita@its-indonesia.com

Overview:
 DNA Extraction
 PCR
 Troubleshooting

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DNA Extraction
Molds  Extraction/Precipitation Method
possess some characteristics of plants, such as
 Enhanced Method
stalk­like structures that resemble plant stems,  Adsorption Chromatography Method
cell walls (though the cell wall of a mold is made
of a polysaccharide called chitin, while the cell
wall of a plant is made of cellulose)

DNA Extraction Requirements Getting prepared

 Disruption of cell wall and membrane to liberate cellular
components.
 Inactivation of DNA­and RNA­degrading enzymes
(DNases, RNases).
 Separation of nucleic acids from
components.
 Extraction/Precipitation method
 Adsorption Chromatography method
   Creating a Nuclease­Free Environment   
Living organism produce several enzymes designed to
degrade DNA molecules. There are several things that you
can do to minimize the risk of exposing your samples to
external DNases.
other cellular  use the autoclaved product (tips, tubes, pestle, etc.)
 work efficient and effectively
 be focus!

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Step 1: Disruption of cell walls by grinding Step 1+2: mechanical disruption and
homogenization in extraction buffer
Extraction/Precipitation method
 Lysis buffer
 Phenol
 Chloroform Grind sample into a fine powder to
 Isoamyl alcohol shear cell walls and membranes

 TE Buffer
Step 2: Lysis of cells in extraction buffer
A homogenizer allows cells to be
mechanically disrupted within the
extraction buffer

Lysis buffer Detergents

sample
Nucleophytopure
resin
Purposes: Chaotropic salts

1. Dissolve cellular membranes Reagen I Reagen II

Metal chelators

Salts
2. Inactivation of DNase
Reducing agents cold box

3. Assist in the removal of contaminants CTAB

(Detergents, Salt+alcohol 70 % alcohol
PVP chaotropic salts,
Plasma membrane
(phospholipid bilayer)
metal chelators,salts,
reducing agents, CTAB) Nucleon™ PhytoPure™
Genomic DNA Extraction Kits
+

3

Adsorptive Chromatography Method
a. Using 260 nm wave length
b. Using 270 nm wave length
c. Using 280 nm wave length
d. Using 230 nm wave length
e. Using 330 nm wave length
3/24/2011
DNA purity and concentration
Hydration shell
Without the chaotropic
agent, DNA wont be able
to join with the membran
When chaotropic agent is
used, hydration shell will
join with it so DNA can
join the membran
(hidrogen bond)
• Pure DNA sample:
260/280 = 1.8 – 2.0
Usefulness
• Contamination by RNA:  Check the DNA purity
260/280 = > 2.0
• Contamination by others:  Check the DNA concentration
260/280 = , 1.8
• Pure DNA sample:
260/270 = around 1.2
• Contamination by phenolate
ion, thiocyanates, others:
PCR
260/230 = 2
• Pure DNA sample:
260/230 = around 1.8
• Pure DNA sample:  Low purity is bad
320 = around 0  Too high concentration is bad  PCR inhibit DNA
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What is PCR
 It’s a means of selectively amplifying a particular segme
nt of DNA.
 The segment may represent a small part of a large and c
omplex mixture of DNAs: e.g. a specific gene of interest
 It can be thought of as a molecular photocopier.

The Basic of PCR Cycling What’s in The Reaction?

30–35 cycles each comprising:
•denaturation (95 denaturation (95° C), 30 sec
•annealing (55–60°), 30 sec.
•extension (72 °C),
time depends on product size.
 Template DNA
 Reaction buffer (Tris, ammonium ions (and/or potassi
um ions), magnesium ions, bovine serum albumin)
 Nucleotides (dNTPs)
 Primers
 DNA polymerase (usually Taq)

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PCR Animations How many copies?

 No target products are made until the third cycle.
 The accumulation is not strictly a doubling at each
cycle in the early phase.
 At 30 cycles there are 1,073,741,764 target copies
(~1 x 109)
 There are also 60 other DNA copies.

How many cycles? Optimizing the PCR Process

 Increasing the cycle number above ~35 has little  Annealing temperature of the primer
positive effect.
 The plateau occurs when:  Mg 2+ concentration
 The reagents are depleted
Accomplished by PureTaq PCR Beads
 The products re­anneal
 Taq polymerase
 The polymerase is damaged
 Unwanted products are accumulate.

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Troubleshooting
TERIMA KASIH
 Use the suitable annealing temperature  gradient PCR
 Make sure that ethanol is completely gone  inhibits PCR
ITS Science Indonesia
 Adding more template can in some cases decrease the Jl. Bukit Gading Raya Blok D/ 9
amount of PCR product obtained. However, since Kelapa Gading, Jakarta ­ 14240, Indonesia
Tel : +62­21­4516222
inhibitors often enter the PCR reaction via the template
preparation, decreasing template volume may improve
PCR results indirectly by reducing the quantity of
inhibiting contaminations
 Typical starting amounts for genomic DNA template are 25­
1000 ng in a 25 µl PCR reaction