Lect-xii & Xiii Pcr And Qpcr.ppt

ANALYSIS OF TRANSGENIC using PCR AND QPCR

Polymerase chain reaction is a method for Amplifying DNA sequences using an which requires

Primers

Which bind to

may be

Random oligonucleotides

DNA template

which is amplified by

Thermostable DNA polymerase

prepared from

Gene-specific primers

Genomic DNA

derived from

Denaturation at > 90 °C

mRNA

Taq polymerase

Primer binding

requires

such as

followed by Known DNA sequence

which runs a cycle of

such as

cDNA synthesis

Amino acid sequence data

Automated thermal cycler

amplification of cDNA

Copying of DNA strands by a polymerase

Polymerase chain reaction (PCR) Amplifying spesifc DNA sequence Polymerase chain reaction (PCR) was invented by Kary Mullis. Kary Mullis won Nobel prize in chemistry in 1993.

THE PRINCIPLE OF PCR PCR components 1. Template DNA DNA containing sequence for amplification 2. Primer • Oligonucleotide • Providing free 3’OH to amplification

start

• bordering the fragment to be amplified. DNA

3. DNA polymerase • DNA polymerase • Amplifying DNA fragment 4. dNTP • Providing DNA bases • dATP, dTTP, dGTP, dCTP

SIKLUS DAN TAHAPAN PCR  DENATURATION (95◦C)  Duplex DNA is heat denatured to give single strands  ANNEALING (55◦C)  two oligonucleotide primers are annealed to their complementary sequences on the target DNA.  EXTENSION (72◦C)  Taq polymerase (thermostable) is used to synthesise complementary strands from the template strands by primer extension. LO 64: menjelaskan prinsip dasar PCR

How to test for GMOs

Test for GMOs by PCR:

1. Grind food 2. Extract DNA from sample 3. Test sample DNA for viable plant DNA 4. Test sample DNA for genetic modifications

• Bio-Rad certified non-GMO food – Verify PCR is not contaminated

Kit Controls

• GMO positive control DNA – Verify GMO-negative result is not due to PCR reaction not working properly • Primers to universal plant gene (Photosystem II)

– Verify viable DNA was extracted

Why amplify a plant gene?

To confirm that viable DNA was extracted and that negative GM result isn’t due to a non-viable template. Use highly conserved chloroplast gene from Photosystem II – part of the light reaction of photosynthesis.

Why use CaMV 35S and NOS?

CaMV 35S – Sequence for the promoter of 35S transcript of the Cauliflower mosaic virus. Used because it functions in every plant cell NOS- Sequence for nopaline synthase terminator from soil bacterium Agrobacterium tumefacians Used because it evolved to be recognized in most plants

Analysis of Results

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GMO positive

1: non-GMO food with plant primers

2: non-GMO food with GMO primers 3. Test food with plant primers

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4: Test food with GMO primers 5: GMO positive template with plant primers 6: GMO positive template with GMO primers 7: PCR MW Ruler

GMO negative

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PCR and False Positives

Transgenic plant produced from Agrobacterium-mediated transformation

Genomic DNA

• In T0 plants, Agrobacterium left over from the initial transformation may still be present in the plant tissue. • Contamination of the genomic DNA with the initial transformation vector that is still present in the agrobacterium can produce a PCR band.

Varian PCR 1. Reverse transcriptase PCR (RT-PCR)  PCR using mRNA as a template  Used to determine the level of gene expression  requires reverse transcriptase

 Oligo (dT) Primer was used to synthesize first strand cDNA.

 PCR primer is used after the first strand cDNA synthesis

Fig. 7.4 RT-PCR. (a) Reverse transcriptase is used to synthesise a cDNA copy of the mRNA. In this example oligo(dT)-primed synthesis is shown. (b) The cDNA product is amplified using genespecific primers. The initial PCR synthesis will copy the cDNA to give a duplex molecule, which is then amplified in the usual way. In many kits available for RT-PCR the entire procedure can be carried out in a single tube.

Processing PCR product PCR products (amplicons) can be:

 analyzed using gel electrophoresis  identified using blotting techniques or

hybridization  cloned into the expression vector

 cloned into a vector (vector-T)  sequenced

PCR quantitative

What is Real-Time PCR? The Polymerase Chain Reaction (PCR) is a process for the amplification of specific fragments of DNA. Real-Time PCR a specialized technique that allows a PCR reaction to be visualized “in real time” as the reaction progresses. As we will see, Real-Time PCR allows us to measure minute amounts of DNA sequences in a sample!

What is Real-Time PCR used for? Real-Time PCR has become a cornerstone of molecular biology: • Gene expression analysis – Cancer research – Drug research • Disease diagnosis and management – Viral quantification • Food testing – Percent GMO food • Animal and plant breeding – Gene copy number

What is Real-Time PCR?

Differences with normal PCR? • 20ul PCR reactions • SYBR Green or probes    

94°C 4 min 94°C 15 sec 40x 61°C 30 sec 72°C 30 sec

Real-time Principles •based on the detection and quantitation of a fluorescent reporter

•In stead of measuring the endpoint we focus on the first significant increase in the amount of PCR product. • The time of the increase correlates inversely to the initial amount of DNA template

Measuring DNA: Ethidium Bromide Ethidium Bromide

http://www.web.virginia.edu/Heidi/chapter12/chp12.htm

Measuring DNA: SYBR Green I SYBR Green I

Ames test results from Molecular Probes Singer et al., Mutat. Res. 1999, 439: 37- 47

RT-PCR • • • •

Isolate RNA from tissues of interest Eliminate all DNA from a sample Make cDNA from mRNA Perform PCR on sample using transgene-specific primers

Real-time PCR or Quantitative PCR • Real-time PCR uses fluorescence as an output for DNA amplification in real-time • The amount of starting template DNA (or cDNA for RNA measurement (real-time RTPCR) is correlated with the Ct number • More DNA = lower Ct; Ct is the cycle number when a threshold amount of DNA is produced during the PCR experiment

http://www.rt-pcr.com/

http://www.youtube.com/watch?v=QVeVIM1yRMU

Advantages of qRT-PCR over RT-PCR?

Q

R

5’

3’

Extension

R Q

Fluorescent Dyes in PCR

Taq

5’

3’

R Q

Hydrolysis

Taq

5’

5’

3’

Taq

Probes

R

5’ 5’

3’

l Taq

R

Signal 5’

5’

3’

What’s Wrong With Agarose Gels? Low sensitivity  Low resolution  Non-automated  Size-based discrimination only  Results are not expressed as numbers  based on personal evaluation  Ethidium bromide staining is not very quantitative  End point analysis 

Imagining Real-Time PCR

So… if YOU started with FOUR times as much DNA template as I did… Then you’d reach 1,000,000 copies exactly TWO cycles earlier than I would! 5000000

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Measuring Quantities

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The “ct value”

Imagining Real-Time PCR

• •

The value that represents the cycle number where the amplification curve crosses an arbitrary threshold. Ct values are directly related to the starting quantity of DNA, by way of the formula:

Quantity = 2^Ct

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Ct Values:

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Measuring Quantities

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Threshold

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 real-time PCR looking for the exact amount of a target sequence or gene in the sample.  During the PCR reaction, you measure its progress by accumulation of a fluorescent signal during amplification.  the Ct, or “threshold cycle.” This spot shows the number of cycles it took to detect a real signal from your samples.  Any real-time PCR run will have many of these curves from several samples, and therefore many Ct values.

threshold

Ct

• Ct values are inverse to the amount of nucleic acid that is in the sample, and correlate to the number of copies in your sample. • Lower Ct values indicate high amounts of targeted nucleic acid, • while higher Ct values mean lower (and even too little) amounts of your target nucleic acid. • Typically, Ct values below 29 cycles show abundant nucleic acids, and Ct values above 38 cycles indicate minimal amounts, and possibly an infection or environmental contamination.

Imagining Real-Time PCR

There’s a DIRECT relationship between the starting amount of DNA, and the cycle number that you’ll reach an arbitrary number of DNA copies (Ct value). DNA amount = 2 ^ Cycle Number C o p y N u m b e r v s. C t - St a n d a r d C u r v e

40

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y =

R

25

Ct

Measuring Quantities

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-3 . 3 1 9 2 x +

2

=

3 9 .77 2

0 .9 9 6 7

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L o g o f c o p y n u m b e r (1 0 )

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What Type of Instruments are used with RealTime PCR?

Real-time PCR instruments consist of TWO main components: • Thermal Cycler (PCR machine) • Optical Module (to detect fluorescence in the tubes during the run)

Quantification and Normalization

• First basic underlying principle: every cycle there is a doubling of product. Quantification and Normalization

• Second basic principle: we do not need to know exact quantities of DNA, instead we will only deal with relative quantities.

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• Third basic principle: we have to have not only a “target” gene but also a “normalizer” gene. • Key formula: Quantity = 2 ^ (Cta – Ctb)

Standard Curve Quantification and Normalization 5000000

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Prepare a 2-fold serial dilution of a DNA sample:

Recomendation: add always a standard curve in every run

“normalizer” gene Quantification and Normalization

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• Knowing the amount of mRNA in one sample from one specific gene does not tell us much.. • You need to know the total amount of mRNA in your sample • You also dont know how much the mRNA level has changed compared to other mRNA levels • Example: mRNA levels of a gene increase 2x after induction

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It is possable that all (1) genexpression in the cell has increased (2) the induced samples contained more total mRNA We

have to compare the expression of our gene to another gene which expression is normally constant, a housekeeping gene (ex. TBP, 18S)

ΔΔCt method experiment

control

-[(Cttg-Ctcg)-(Cttg-Ctcg)]

2

Ex!  Ct = target gene– ref gene  Ct = 9.70  Ct = target gene– ref gene  Ct = -1.70 Difference = Ct-Ct = Ct = 9.70-(-1.7) = 11.40 Fold change = 211.40 = 2702

Always in duplicate or triplicate!