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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