pcr ppt presentation

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AP®︎/College Biology

Course: ap®︎/college biology   >   unit 6.

• Introduction to genetic engineering
• Intro to biotechnology
• DNA cloning and recombinant DNA
• Overview: DNA cloning

Polymerase chain reaction (PCR)

• Gel electrophoresis
• DNA sequencing
• Applications of DNA technologies
• Biotechnology

Key points:

• Polymerase chain reaction , or PCR , is a technique to make many copies of a specific DNA region in vitro (in a test tube rather than an organism).
• PCR relies on a thermostable DNA polymerase, Taq polymerase , and requires DNA primers designed specifically for the DNA region of interest.
• In PCR, the reaction is repeatedly cycled through a series of temperature changes, which allow many copies of the target region to be produced.
• PCR has many research and practical applications. It is routinely used in DNA cloning, medical diagnostics, and forensic analysis of DNA.

What is PCR?

Taq polymerase, pcr primers, the steps of pcr.

• Denaturation ( 96 ° C ‍   ): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step.
• Annealing ( 55 ‍   - ‍   65 ‍   ° C ‍   ): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.
• Extension ( 72 ° C ‍   ): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.

Using gel electrophoresis to visualize the results of PCR

Applications of pcr, sample problem: pcr in forensics.

• (Choice A)   Suspect 1 ‍   A Suspect 1 ‍  
• (Choice B)   Suspect 2 ‍   B Suspect 2 ‍  
• (Choice C)   Suspect 3 ‍   C Suspect 3 ‍  
• (Choice D)   None of the suspects D None of the suspects
• Crime scene DNA: homozygous 200 ‍   bp allele
• Suspect 1 ‍   : homozygous 300 ‍   bp allele
• Suspect 2 ‍   : heterozygous
• Suspect 3 ‍   homozygous 200 ‍   bp allele

More about PCR and forensics

Attribution:, works cited:.

• Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., and Jackson, R. B. (2011). Forensic evidence and genetic profiles. (10th ed., pp. 430-431). San Francisco, CA: Pearson.

References:

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Polymerase chain reaction (interactive).

• Description

Polymerase chain reaction (PCR) enables researchers to produce millions of copies of a specific DNA sequence in approximately two hours. This automated process bypasses the need to use bacteria for amplifying DNA.

This animation is featured in our "Spotlight Collection" on Polymerase Chain Reaction, along with video interviews with Kary Mullis, a 3D molecular animation of PCR, and several laboratory protocols.

This animation is also available as VIDEO .

Polymerase chain reaction (PCR) enables researchers to produce millions of copies of a specific DNA sequence in approximately two hours. This automated process bypasses the need to use bacteria for amplifying DNA. This animation is featured in our "Spotlight Collection" on Polymerase Chain Reaction, along with video interviews with Kary Mullis, a 3D molecular animation of PCR, and several laboratory protocols.

polymerase chain reaction,dna polymerase,dna sequence,automated process,chain reaction,bacteria

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15625. polymerase chain reaction (pcr).

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• Source: DNAi

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Kary Mullis explains how the polymerase chain reaction (PCR) was named.

15624. Kary Mullis

Image of Kary Mullis. In 1985, Kary Mullis invented the polymerase chain reaction (PCR), a method of amplifying or producing many copies of a specific piece of DNA. The revelation came to this eccentric character on a drive in northern California.

15140. Making many DNA copies, Kary Mullis

Kary Mullis talks about his discovery of the polymerase chain reaction (PCR), a process that allows chemists to produce many copies of a specific fragment of DNA.

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The quartz wafer is in the holding position on the DNA synthesizer. The wafer is moved to a vertical reaction vessel for the process of DNA chain elongation.

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• Source: DNALC.DNAFTB

16515. Animation 23: A gene is a discrete sequence of DNA nucleotides.

Fred Sanger outlines DNA sequencing.

15139. Finding DNA to copy, Kary Mullis

Kary Mullis speaks about the process of find a specific fragment of DNA amongst many pieces in a complex mixture.

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Polymerase Chain Reaction (PCR)- Principle, Procedure, Types, Applications and Animation

Polymerase Chain Reaction (PCR) is a powerful method for amplifying particular segments of DNA, distinct from cloning and propagation within the host cell. This procedure is carried out entirely biochemically, that is, in vitro. PCR was invented by Kary Mullis in 1983. He shared the Nobel Prize in chemistry with Michael Smith in 1993.

Principle of PCR

PCR uses the enzyme DNA polymerase that directs the synthesis of DNA from deoxynucleotide substrates on a single-stranded DNA template. DNA polymerase adds nucleotides to the 3` end of a custom-designed oligonucleotide when it is annealed to a longer template DNA. Thus, if a synthetic oligonucleotide is annealed to a single-stranded template that contains a region complementary to the oligonucleotide, DNA polymerase can use the oligonucleotide as a primer and elongate its 3` end to generate an extended region of double stranded DNA.

Procedure/Steps of PCR

1. Denaturation

The DNA template is heated to 94° C. This breaks the weak hydrogen bonds that hold DNA strands together in a helix, allowing the strands to separate creating single stranded DNA.

2. Annealing

The mixture is cooled to anywhere from 50-70° C. This allows the primers to bind (anneal) to their complementary sequence in the template DNA.

3. Extension

The reaction is then heated to 72° C, the optimal temperature for DNA polymerase to act. DNA polymerase extends the primers, adding nucleotides onto the primer in a sequential manner, using the target DNA as a template.

With one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA. These two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the template is increased exponentially.

Types of PCR

• Real-time PCR
• Quantitative real time PCR (Q-RT PCR)
• Reverse Transcriptase PCR (RT-PCR)
• Multiplex PCR
• Long-range PCR
• Single-cell PCR
• Fast-cycling PCR
• Methylation-specific PCR (MSP)
• Hot start PCR
• High-fidelity PCR
• In situ PCR
• Variable Number of Tandem Repeats (VNTR) PCR
• Asymmetric PCR
• Repetitive sequence-based PCR
• Overlap extension PCR
• Assemble PCR
• Intersequence-specific PCR(ISSR)
• Ligation-mediated PCR
• Methylation –specifin PCR
• Miniprimer PCR
• Solid phase PCR
• Touch down PCR, etc

Applications of PCR

• PCR is used in analyzing clinical specimens for the presence of infectious agents, including HIV, hepatitis, malaria, anthrax, etc.
• PCR can provide information on a patient’s prognosis, and predict response or resistance to therapy. Many cancers are characterized by small mutations in certain genes, and this is what PCR is employed to identify.
• PCR is used in the analysis of mutations that occur in many genetic diseases (e.g. cystic fibrosis, sickle cell anaemia, phenylketonuria, muscular dystrophy).
• PCR is also used in forensics laboratories and is especially useful because only a tiny amount of original DNA is required, for example, sufficient DNA can be obtained from a droplet of blood or a single hair.
• PCR is an essential technique in cloning procedure which allows generation of large amounts of pure DNA from tiny amount of template strand and further study of a particular gene.
• The Human Genome Project (HGP) for determining the sequence of the 3 billion base pairs in the human genome, relied heavily on PCR.
• PCR has been used to identify and to explore relationships among species in the field of evolutionary biology. In anthropology, it is also used to understand the ancient human migration patterns. In archaeology, it has been used to spot the ancient human race. PCR commonly used by Paleontologists to amplify DNA from extinct species or cryopreserved fossils of millions years and thus can be further studied to elucidate on.

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23 thoughts on “Polymerase Chain Reaction (PCR)- Principle, Procedure, Types, Applications and Animation”

What about the tetra ARMS PCR technique?

i just love the way that PCR is explained. its to easy to understand. thanks for this

Best principle of PCR ever explained. Thank you !!!!

I was started working on PCR after finishing my Master, loved the way it produce the accurate and fast result. I was running 3 to 4 test at a time and it gives amazing result. With the help of PCR result it become easy to find how much infection in the blood and based on infection the Specialist doctor can treat the patient and the strength of medicine can be given to cure fastest. Thanks to those people who found this way of doing.

Hi Sir!One important question. How the PCR is working in microbial diagonistics in medical.

specific genetic regions of different microbs are detected and amplified with the help of known primers.

Dear All, I am a medical Microbiologist. I’m interested in Post doctorate fellowship research into genetic studies of Multi-Drug Resistant Tuberculosis. Can somebody link me up. Thanks

how can this polymerase chain reaction be applied in the detection of zika virus in the lab ?

what are u doing explain ur work lil briefly

simply PCR work in 3 steps they are Denaturation where the dubble strand DNA break into single strand. Its temperature is 94 degree Celsius, than annealing temperature where primer bind it temp generally 50-70 degree Celsius and finally extension where the Taq polymerase act on the DNA and primer joining them with hydrogen bonding. simply PCR is the machine which give control temperature, where we prepare our reaction mixture which contain 1.Taq buffer 2.Taq polymerase 3. dNTP which provide nucleotide for the new strands 4.Forward and reverse primer for The reverse primer is designed to attach to the complementary strand to synthesize DNA in the reverse direction — towards the forward primer. The primers are added to PCR experiments to initiate the process of replication by providing the initial nucleotides to the new strand. 5. Template DNA 6. HPLC graded water.

I am working on a bacteria specie with intention of isolating it from different sources and comparing their antibiotic resistance plasmid content and other characteristics.How do I determine the right primers to use for my work?

First i most comment your effort you’re doing great. I am writting to see if anyone can link me to a reputable university where i can run P.hD in molecular microbiology with MRSA as my microorganism of interest. I worked on it in my Masters and i want to continue with it. Am waiting for any assistance one can render.

wat s d role of Thermus aquaticus in PCR and how does it works?

It’s a thermo stable bacterium (Thermus aguatics ) to maintain a temperature duringPCR

An enzyme DNA polymerase is an indispensable tool for DNA replication, we all knows that enzymes are temperature sensitive, Taq DNA is DNA polymerase that’s isolated from bacteria Thermus aquaticus, This enzyme can withstand different temperatures 92,54, 72 degrees.. If during amplification of ur desired DNA (wether Replication or PCR) ur polymerase became inactivated by heat, then the process adding nucleotides and subsequent elongation stops. Therefore Taq DNA is the suitable enzyme to used.

thermus aquaticus is a bacterial specie from which the polymerase enzyme can be obtaines which is use for polymerization in pcr

The taq polymerase used for PCR is from Thermus Aquaticus. And it does not “maintain a tempreature during PCR” but, tolerates and survives in high tempreature, unlike most other bacteria. PCR uses high tempreatures and these polymerases hence work effectively.

Awesome info. Thanks! It would be nice if you share the distinctive futures of the specific types of PCR too. Kind regards

Hi Sir!One important question. How the PCR is working in microbial diagonistics in medical. I need a perfect to present a seminar so plzzz reply me “AS SOON AS POSSIBLE”. “IAM EXPECTING MORE FROM THIS WEBSITE”

1st analyse the microbe DNA and get sequence and then identified the microbe specious then proceed for antimicrobial methods.

dr how can downlaod the books of immunology?

The multiplex endpoint PCR technology offers a number of potential advantages, results are available in a matter of hours rather than days, the extreme sensibility facilitates detection of even minutes the amounts of pathogen DNA in clinical samples. While microbiological culture is likely to remain a gold standard for infection diagnosis, there is growing interest at the potential of PCR technology to provide early, time critical information based on detection and recognition of bacterial or fungal pathogen DNA. By this new modern method, the results are chosen in perquisite for giving adequate antibiotics treatment as early as possible in order to improve the standard of care. Dr. Aurel

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Polymerase chain reaction (PCR) - PowerPoint PPT Presentation

Polymerase chain reaction (PCR)

Polymerase chain reaction (pcr) is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. these include dna cloning for sequencing, dna-based phylogeny, or functional analysis of genes, the diagnosis of hereditary diseases, and the detection and diagnosis of infectious diseases. – powerpoint ppt presentation.

• Table of Contents
• What Is PCR (polymerase chain reaction)?
• What Is the Conventional PCR Principle?
• What Are the Components of a Conventional PCR Setup?
• How Does Conventional PCR Work?
• What Are the Three Basic Steps of Conventional PCR?
• Denaturation step
• Annealing step
• Extension/elongation step
• Applications of PCR
• PCR and Infectious Diseases
• PCR Analysis and COVID-19 Infection Detection
• Create Your Free Account and Try Our Simulation Conventional PCR
• PCR is based on three simple steps required for any DNA synthesis reaction
• Denaturation of the template into single strands.
• Annealing of primers to each original strand for new strand synthesis.
• Extension of the new DNA strands from the primers.
• These reactions may be carried out with any DNA polymerase and result in the synthesis of defined portions of the original DNA sequence. However, in order to achieve more than one round of synthesis, the templates must again be denatured, which requires temperatures well above those that inactivate most enzymes. Therefore, initial attempts at cyclic DNA synthesis were carried out by adding fresh polymerase after each denaturation step (1,2). The cost of such a protocol becomes rapidly prohibitive.
• Typically, PCR consists of a series of 2040 repeated temperature changes, called cycles, with each cycle commonly consisting of 23 discrete temperature steps. The cycling is often preceded by a single temperature step at a high temperature (gt90 C) and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature of the primers.

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PCR (Polymerase Chain Reaction) PPT: Definition, Principles and Components

PCR (Polymerase Chain Reaction) PPT: Definition, Principles and Components Free Download: It is a modern approach evolved through Kary Mullis withinside the 1980s. PCR is primarily based totally on the use of the capacity of DNA polymerase to synthesize new strand of DNA complementary to the provided template strand.

Because DNA polymerase can upload a nucleotide best onto a preexisting 3′-OH group, it wishes a primer to which it may upload the primary nucleotide. This requirement makes it feasible to delineate a selected location of template collection that the researcher desires to amplify. At the cease of the PCR reaction, the unique collection could be collected in billions of copies (amplicons).

Table of Content

• Introduction
• Principle of PCR
• Components of PCR
• Types of PCR
• Steps of PCR
• Applications of PCR
•  Conclusion

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49 Best Pcr-Themed Templates for PowerPoint & Google Slides

With over 6 million presentation templates available for you to choose from, crystalgraphics is the award-winning provider of the world’s largest collection of templates for powerpoint and google slides. so, take your time and look around. you’ll like what you see whether you want 1 great template or an ongoing subscription, we've got affordable purchasing options and 24/7 download access to fit your needs. thanks to our unbeatable combination of quality, selection and unique customization options, crystalgraphics is the company you can count on for your presentation enhancement needs. just ask any of our thousands of satisfied customers from virtually every leading company around the world. they love our products. we think you will, too" id="category_description">crystalgraphics creates templates designed to make even average presentations look incredible. below you’ll see thumbnail sized previews of the title slides of a few of our 49 best pcr templates for powerpoint and google slides. the text you’ll see in in those slides is just example text. the pcr-related image or video you’ll see in the background of each title slide is designed to help you set the stage for your pcr-related topics and it is included with that template. in addition to the title slides, each of our templates comes with 17 additional slide layouts that you can use to create an unlimited number of presentation slides with your own added text and images. and every template is available in both widescreen and standard formats. with over 6 million presentation templates available for you to choose from, crystalgraphics is the award-winning provider of the world’s largest collection of templates for powerpoint and google slides. so, take your time and look around. you’ll like what you see whether you want 1 great template or an ongoing subscription, we've got affordable purchasing options and 24/7 download access to fit your needs. thanks to our unbeatable combination of quality, selection and unique customization options, crystalgraphics is the company you can count on for your presentation enhancement needs. just ask any of our thousands of satisfied customers from virtually every leading company around the world. they love our products. we think you will, too.

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Real-Time PCR!

Apr 30, 2012

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Real-Time PCR!. Fundamentals of Real-Time PCR. What is Real-Time PCR?. Real-Time PCR a specialized technique that allows a PCR reaction to be visualized “in real time” as the reaction progresses. This enables researchers to quantify the amount of DNA in the sample at the start of the reaction!.

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Real-Time PCR! Fundamentals of Real-Time PCR

What is Real-Time PCR? Real-Time PCR a specialized technique that allows a PCR reaction to be visualized “in real time” as the reaction progresses. This enables researchers to quantify the amount of DNA in the sample at the start of the reaction!

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 • Viral quantification • Food testing • Percent GMO food • Transgenic research • Gene copy number

How does real-time PCR work? To best understand what real-time PCR is, let’s review how regular PCR works...

5’ 3’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 3’ 5’ 3’ The Polymerase Chain ReactionHow does PCR work?? d.NTPs Primers Thermal Stable DNA Polymerase Add to Reaction Tube Denaturation Annealing

5’ 5’ 5’ 3’ 3’ 3’ 5’ 5’ 3’ 3’ 3’ 3’ 5’ 5’ 3’ Taq Taq 5’ 5’ 5’ Taq Taq 5’ The Polymerase Chain ReactionHow does PCR work?? Extension Extension Continued Repeat

3’ 3’ 5’ 5’ 5’ 3’ 3’ 3’ 5’ 3’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ The Polymerase Chain ReactionHow does PCR work?? Cycle 2 4 Copies Cycle 3 8 Copies

Imagining Real-Time PCR …So that’s how PCR is usually presented. To understand real-time PCR, let’s imagine ourselves in a PCR reaction tube at cycle number 25…

Imagining Real-Time PCR What’s in our tube, at cycle number 25? A soup of nucleotides, primers, template, amplicons, enzyme, etc. 1,000,000 copies of the amplicon right now.

Imagining Real-Time PCRHow did we get here? What was it like last cycle, 24? Almost exactly the same, except there were only 500,000 copies of the amplicon. And the cycle before that, 23? Almost the same, but only 250,000 copies of the amplicon. And what about cycle 22? Not a whole lot different. 125,000 copies of the amplicon.

Imagining Real-Time PCRHow did we get here? If we were to graph the amount of DNA in our tube, from the start until right now, at cycle 25, the graph would look like this:

Imagining Real-Time PCRHow did we get here? ? So, right now we’re at cycle 25 in a soup with 1,000,000 copies of the target. What’s it going to be like after the next cycle, in cycle 26?

Imagining Real-Time PCRSo where are we going? What’s it going to be like after the next cycle, in cycle 26? Probably there will be 2,000,000 amplicons. And cycle 27? Maybe 4,000,000 amplicons. And at cycle 200? In theory, there would be 1,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000 amplicons… Or 10^35 tonnes of DNA… To put this in perspective, that would be equivalent to the weight of ten billion planets the size of Earth!!!!

Imagining Real-Time PCRSo where are we going? A clump of DNA the size of ten billion planets won’t quite fit in our PCR tube anymore. Realistically, at the chain reaction progresses, it gets exponentially harder to find primers, and nucleotides. And the polymerase is wearing out. So exponential growth does not go on forever!

Imagining Real-Time PCRSo where are we going? If we plot the amount of DNA in our tube going forward from cycle 25, we see that it actually looks like this:

Imagining Real-Time PCRMeasuringQuantities How can all this be used to measure DNA quantities?? What if YOU started with FOUR times as much DNA template as I did? I have 1,000,000 copies at cycle 25. You have 4,000,000 copies! So… You had 2,000,000 copies at cycle 24. And… You had 1,000,000 copies at cycle 23.

Imagining Real-Time PCRMeasuringQuantities 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!

Imagining Real-Time PCRMeasuringQuantities What if YOU started with EIGHT times LESS DNA template than I did? You’d only have 125,000 copies right now at cycle 25… …and you’ll have 250,000 at 26, 500,000 at 27, and by cycle 28 you’ll have caught up with 1,000,000 copies! So… you’d reach 1,000,000 copies exactly THREE cycles later than I would!

Imagining Real-Time PCRMeasuringQuantities We describe the position of the lines with a value that represents the cycle number where the trace crosses an arbitrary threshold. This is called the “Ct Value”. Ct values are directly related to the starting quantity of DNA, by way of the formula: Quantity = 2^Ct Ct Values: 25 23 28

Imagining Real-Time PCRMeasuringQuantities 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

How do We Measure DNA in a PCR Reaction? We use reagents that fluoresce in the presence of amplified DNA! Ethidium bromide and SYBR Green I dye are two such reagents. They bind to double-stranded DNA and emit light when illuminated with a specific wavelength. SYBR Green I dye fluoresces much more brightly than ethidium.

5’ 5’ 5’ 3’ 3’ 3’ 5’ 5’ 3’ 3’ 3’ 3’ 5’ 5’ 3’ Taq Taq 5’ 5’ 5’ Taq Taq l l l ID ID ID ID ID ID ID ID ID ID 5’ l l How do We Measure DNA in a PCR Reaction? Extension Apply Excitation Wavelength Repeat

What Type of Instruments are used with Real-Time 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)

What Type of Instruments are used with Real-Time PCR? An example of such an instrument is the Bio-Rad iQ5 real-time PCR instrument.

Real-Time PCRActual Data • This is some actual data from a recent real-time PCR run. • Data like this can easily be generated by preparing a dilution series of DNA. Color key: Green=100X, Red=10000X, Blue=1000000X , Black=NTC.

Real-Time PCR – the Concept of MELT CURVES… • Melt curves can tell us what products are in a reaction. RFU vs T dRFU/dT

Real-Time PCRThe Concept of MELT CURVES • Different amplicons will have different melt peaks. • Primer-Dimers will have a very different melt peak. Color key: Green=100X, Red=10000X, Blue=1000000X , Black=NTC.

GMO Investigator Kit Can we extend the GMO Kit to demonstrate Real-Time PCR???

GMO Investigator Kit contents • Bio-Rad certified Non-GMO food • InstaGene • Master Mix • GMO primers • Plant PSII primers • GMO & PSII positive control DNA • PCR MW Ruler • DPTPs, microtubes, PCR tubes, foam floats • Manual • Not Included but required: • Thermal cycler • Water bath/heat block • Electrophoresis Module (agarose, TAE buffer & Fast Blast DNA stain) • Electrophoresis equipment & power supply • 2-20 ul pipettes & barrier tips

GMO Investigator Kitin Real-Time • To run the GMO Kit in real-time, only two additional items are needed… • iQ SYBR Green Supermix • A real-time PCR instrument

Control DNA Dilutions, Plant Primer GMO Investigator Kitin Real-Time

Control DNA Dilutions, GMO Primer GMO Investigator Kitin Real-Time

GMO Investigator Kitin Real-Time Spiked Bean Samples Color key: black=NTC, green=plant primers, red=GMO primers. Curves represent pure DNA and DNA-spiked plant samples.

A Few Food Samples… GMO Investigator Kitin Real-Time Color key: black=NTC, green=plant primers, red=GMO primers. Order of curves left to right = negative control beans, tortilla chips, soy sausage, corn nuts.

A Few Food Samples, Melt Curve GMO Investigator Kitin Real-Time Color key: black=NTC, green=plant primers, red=GMO primers. Order of peak heights (highest to lowest) is negative control bean, tortilla chips, and sausage.

A Few Food Samples, Gels GMO Investigator Kitin Real-Time Key: Lane 1=NTC; 2 = positive control DNA 1:1000; 3 = positive control DNA 1:10,000; 4 = non-GMO beans spiked with #2; 5 = non-GMO beans spiked with #3; 6 = non-GMO beans; 7 = tortilla chips; 8 = corn nuts; 9 = soy sausage. Each DNA sample loaded in pairs, plant primers on left, GMO primers on right. Ladders on end are 1000,750,500 and 200bp. Upper row consists of PCR reactions carried out exactly according to kit instructions using kit reagents. Lower row consists of same with exception of iQ SYBR Green substituted for kit supermix.

Classroom Results, Contra Costa College May 2006 GMO Investigator Kitin Real-Time Plant GMO Color key: blue = pure GMO DNA, pink = papaya, red = corn chips, green = tofu, brown = soy flour, purple = non-GMO beans, black = no template control.

Classroom Results, Contra Costa College May 2006 GMO Investigator Kitin Real-Time GMO Plant

GMO Investigator Kit Quantification and Normalization

Quantification and Normalization • First basic underlying principle: every cycle there is a doubling of product. • Second basic principle: we do not need to know exact quantities of DNA, instead we will only deal with relative quantities. • Third basic principle: we have to have not only a “target” gene but also a “normalizer” gene. • Key formula: • Quantity = 2 ^ (Cta – Ctb)

Quantification and Normalization • Example: Ct values for the plant gene and GMO gene from different food samples.

Quantification and Normalization • First we’ll compare relative amounts of plant DNA. • Using the formula that relates relative quantity to 2^(Cta-Ctb), we can calculate amounts of DNA relative to a single sample.

Quantification and Normalization • Second we’ll determine relative amounts of GMO DNA… • We can do the exact same calculations for the Ct values from the GMO genes of the same food samples.

Quantification and Normalization • Now we compare the amount of plant to GMO DNA in each sample… • This is called delta-delta-Ct analysis, and is the basis of real-time quantification analysis.

Summary Quantification and Normalization • First we determined how much plant DNA is in each sample, • Second we determined how much GMO DNA is in each sample, • Finally we corrected / normalized the GMO DNA against the plant DNA to determine relative amount of GMO for each sample.

Quantification and Normalization • Practice! Try this example. • What is the GMO content of product A and product B??

Quantification and Normalization • Results… • What is the GMO content of product A and product B??

Real-TimeDemonstration Real-Time PCR Demonstration

Real-TimeDemonstration Experimental Protocol • Standard curve with serial 10-fold dilutions • Two replicates on standard curve • Multiple unknown samples • 25ul reactions • iQ SYBR Green Supermix (2x) • 94C 3 min • 94C 30 sec • 59C 60 sec • Goto Step 2 40 Times

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