Molecular methods for food safety testing
POLYMERASE CHAIN REACTION
(Phản ứng chuỗi trùng hợp)
K. Mullis, 1983
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Watson and Crick, 1953
Basic information Discovery of the DNA
molecule structure
Deoxyribo Nucleic Acid
DNA = nucleotide polymers
Four types of nucleotides: A, T, C and G
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NUCLEOTIDES
Nitrogen
ous base
Phosph
ate
group
Sugar:
pentose
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Nitrogenous bases 5 carbons Oses
RNA DNA
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Erwinn Chargaff (1947)
If you separate a DNA molecule into
nucleotides, you will allways obtain:
A=T
and
There could be more AT than CG pairs or
C=G the contrary (depending on the species),
but there is allways the same proportion of
A and T, and of C and G.
Why ?
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Watson and Crick ’s hypothesis:
A can bind with T and C with G:
A with T: two
hydrogen bonds
(low bonds).
C with G: three
hydrogen bonds
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A human cell contains 46 DNA molecules
Each DNA molecule wind around proteins (histones) and
forms a chromosome.
DNA One chromosome =
one DNA molecule
combined with
proteins.
Histones
All chromosomes
form chromatine
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Melting temperature (Tm) of DNA Hyperchromic effect
The value of optical density will increase
when DNA change from double-strand to
single-strand structure
When a double
To detect the
stranded DNA is
transformation of DNA
heated over
from double-strand to
melting temperature (Tm), single-strand structure:
2 strands will be separated due to measure the change of optical density
breakage of hydrogen bonds at the wavelength of 260nm.
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1. INTRODUCTION OF PCR
Described by K. Mullis et al. in 1983 (Nobel
prize at Chemistry 1993).
Amplification of DNA fragments.
In-vitro reaction by enzyme (DNA
polymerase).
Repeated thermal processes (up to 35-40
cycles), including heating to 92-950C, cooling
to 37-650C and incubation at 720C.
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2. REQUIREMENT
2. REQUIREMENT Primers
short pieces of single-stranded DNA that are
DNA template: complementary base pairing to the target
the sample DNA that contains the target sequence.
sequence. The polymerase begins synthesizing new DNA
At the beginning of the reaction, high from the end of the primer.
temperature is applied to the original double-
stranded DNA molecule to separate the
strands from each other.
Sequencing the ends of target DNA fragments
to design primers.
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2. REQUIREMENT 2. REQUIREMENT
DNA polymerase
A type of enzyme that synthesizes new strands Nucleotides (dNTPs or deoxynucleotide
of DNA complementary to the target sequence. triphosphates)
The first and most commonly used of these Single units of the bases A, T, G, and C, which
enzymes isTaqDNA polymerase (fromThermis are essentially "building blocks" for new DNA
aquaticus), whereasPfuDNA polymerase strands.
(fromPyrococcus furiosus) is used widely With proper primers and at suitable condition
because of its higher fidelity when copying DNA.
for DNA polymerase, DNA fragments will be
These enzymes both have two capabilities
amplified to make a remarkable copies that
suitable for PCR:
they can be observed after staining with
1) they can generate new strands of DNA using
a DNA template and primers ethidium bromide (BET).
2) they are heat resistant. 27 28
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3. PCR PROCESS
PCR is a chain of continuous cycles, each cycle
composed of 3 steps.
After each cycle, 1 template DNA will make 2
new DNA.
Final result of PCR: after n cycles, 2n copies of
double-stranded DNA are made (in theory)
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Step 1 (denaturation): DNA molecule is denatured Step 2 (anealation or hybridization): reduce
at the temperature higher than its Tm, usually at temperature to 40 - 700C depending on
94-95 0C in 30s – 1 min. At this temperature, all primer, usually at 52 - 540C in 30s – 1min.
helix structure of template DNA are separated to At this temperature, 2 primers will bind
make single strands which are template to with 2 DNA strands for the start of DNA
synthesize primers and DNA polymerase. polymerase.
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Step 3 (elongation): increase temperature to
720C (optimum temperature for thermal Taq
DNA polymerase). At this temperature, DNA
polymerase will lengthen the DNA strands.
The materials for this step is 4 types of
dNTPs. The duration for this step ranges
from 30s – several mins.
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4.1 DNA template
4. FACTORS AFFECTING DNA template is the desired DNA
PCR fragment that need to be amplified
The PCR is better on purified DNA
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4.2 Enzyme
4.3 Primers and hybrid temperature
Can work at high temperature.
The sequence of primers are designed so that
Enzyme Taq there is no additional hybrid between 2
polymerase are primers.
usually extracted Tm of 2 primers are not too far away
from thermal Primers must be specific for the amplified
bacterium DNA
Thermus aquaticus The sequence of primers is not too long,
(80-950C) usually not longer than 1 kb.
Thermus aquaticus
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4.4 Other factors 5. SIGNIFICANCE OF PCR
Advantages:
The concentration of 4 types of
Fast (several
hours).
nucleotide: 20-200µM each type.
Simple and cheap (labor, materials,
The concentration of ion Mg2+ to
equipment…)
connect dNTP with enzyme and
activate enzyme polymerase The purity of sample are not very high
Disadvantages:
Number of cycle: depend on the
quantity of DNA template, usually not The length of sequence is short (<1.5kb)
higher than 40 cycles The infection of previous steps
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The faults of Taq polymerase 42
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6. SOME APPLICATIONS OF PCR
Produce probe
Appication in genetic medicine, illness
diagnose, forensis science…
Amplification of RNA
RT – PCR (Reverse Transcriptase –
PCR) RNA cDNA by Reverse
Transcriptase.
cDNA is amplified by Taq
polymerase.
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Sample preparation
50g sample + 450mL alkaline peptone water Incubation 370C, 6h.
(APW) homogenization
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Characterization by agar spreading plates
or biochemical reactions
Catalase positive Catalase negative
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Non-mobile
Mobile
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Định danh phaûn öùng… (tt)
API20E
a. Oxidase test
b. Nitrate reduction
c. Motility
d. Growth on MacConkey agar
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DNA Extraction
Centrifugation
Remove supernatant
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polysaccharides
Mix with 50µL distilled water.
Heat 1100C, 5 mins.
DNA
→ DNA template for PCR.
Plasmids
DNA
degradation
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PCR Master mix
BG61: 5’-GCGAATTCGATAGGGTGTTAACC-3’.
PCR buffer 10X + MgCl 2mM + dNTP 200
BG62: 5’-CGAATCCTTGAACATACGCAGC-3’. μM + Primer 250 mM + Taq DNA polymerase
0.5 UI + 2-times distilled water (enough 50
μL), DNA 1µL.
Number of cycle: 30.
Setting reaction:
Denaturation 940C: 1 min
Anneal 600C: 1 min
0
Extension 72 C: 1 min
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Gel electrophoresis
Run PCR
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Gel electrophoresis Gel electrophoresis
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Gel electrophoresis Gel electrophoresis
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Limitations of PCR and RT-PCR
Types of PCR
The PCR reaction starts to generate copies of the target
sequence exponentially. Only during the exponential phase Multiplex PCR
of the PCR reaction is it possible to extrapolate back to
Long-range PCR
determine the starting quantity of the target sequence
contained in the sample. Single-cell PCR
Because of inhibitors of the polymerase reaction found in the Fast-cycling PCR
sample, reagent limitation, accumulation of pyrophosphate Methylation-specific PCR (MSP)
molecules, and self-annealing of the accumulating product, Hot start PCR
the PCR reaction eventually ceases to amplify target High-fidelity PCR
sequence at an exponential rate and a "plateau effect"
RAPD: Rapid amplified polymorphic DNA analysis
occurs, making the end point quantification of PCR products
unreliable. This is the attribute of PCR that makes Real-Time RACE: Rapid amplification of cDNA ends
Quantitative RT-PCR so necessary. In situ PCR
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Differential display PCR 66
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Quantitative PCR or
Real Time PCR (qPCR)
Allow the estimation of the amount of a given
sequence present in a sample — a technique often
Quantitative PCR or applied to quantitatively determine levels of gene
Real Time PCR (qPCR)
expression.
Quantitative PCR is an established tool for DNA
quantification that measures the accumulation of
DNA product after each round of PCR amplification.
qPCR allows the quantification and detection of a
specific DNA sequence in real time since it measures
concentration while the synthesis process is taking
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Quantitative PCR or Real Time PCR (qPCR) Quantitative PCR or Real Time PCR (qPCR)
An interesting technique combination is real-time
Two methods for simultaneous detection and PCR and reverse transcription
quantification: RT-qPCR: allows the quantification of a small
using fluorescent dyes that are retained quantity of RNA.
nonspecifically in between the double strands. Through this combined technique, mRNA is
using probes that code for specific sequences converted to cDNA, which is further quantified using
and are fluorescently labeled. qPCR.
Detection of DNA using these methods can This technique lowers the possibility of error at the
only be seen after the hybridization of probes end point of PCR, increasing chances for detection
of genes associated with genetic diseases such as
with its complementary DNA takes place.
cancer. Laboratories use RT-qPCR for the purpose
of sensitively measuring gene regulation.
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Quantitative PCR or Real Time PCR (qPCR)
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Pre-enrichment media
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Pre-enrichment media Pre-enrichment media
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Pre-enrichment media Pre-enrichment media
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Pre-enrichment media Pre-enrichment media
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DNA preparation and purification
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Mill DNA extraction
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