what is qpcr and how does it work? - edvotekin 1984, dr. kary mullis revolutionized the ﬁ eld of...

S-54.141008

S-54Edvo-Kit #S-54

What is qPCR and How Does It Work?Experiment Objective:

This experiment explores the principles of qPCR by analyzing colorful dye samples using agarose gel electrophoresis. Students will observe the relationship between cycle number and amount of DNA present within a sample.

See page 3 for storage instructions.

NEW

Page

Experiment Components 3

Experiment Requirements 3

Background Information 4

Experiment Procedures Experiment Overview 7 Agarose Gel Electrophoresis 9 Study Questions 11 Instructor's Guidelines 12 Pre-Lab Preparations 13 Experiment Results and Analysis 14 Study Questions and Answers 15

Appendices 16

Material Safety Data Sheets can be found on our website:www.edvotek.com

EDVOTEK and The Biotechnology Education Company are regis-tered trademarks of EDVOTEK, Inc. Ready-to-Load, QuickStrips and UltraSpec-Agarose are trademarks of EDVOTEK, Inc.

Table of Contents

What is qPCR and How Does It Work? EDVO-Kit S-54

1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com

2

Duplication of any part of this document is permitted for non-profi t educational purposes only. Copyright © 1989-2014 EDVOTEK, Inc., all rights reserved. S-54.141008


Experiment Components

READY-TO-LOAD™ SAMPLES FOR ELECTROPHORESISStore all components at room temperature.

Components (in QuickStrip™ format) Check (√)

A Standard dyes with assigned base pair equivalents ❑B Sample after 10 cycles ❑C Sample after 20 cycles ❑D Sample after 30 cycles ❑E Sample after 40 cycles ❑ REAGENTS & SUPPLIES

• UltraSpec-Agarose™ ❑• Electrophoresis Buffer (50x) ❑• Practice Gel Loading Solution ❑• 1 ml pipet ❑• Microtipped Transfer Pipets ❑

Experiment #S-54 is designed for 10 gels.

The QuickStrip™ samples are stable at room temperature for up to one month after receipt. However, if the experiment will not be con-ducted within this time frame, it is recommended that the QuickStrip™ samples be stored in the refrigerator.

• Horizontal gel electrophoresis apparatus• D.C. power supply• Automatic micropipets with tips• Balance• Microwave, hot plate or burner• Pipet pump• 250 ml fl asks or beakers• Hot gloves• Safety goggles and disposable laboratory gloves• DNA visualization system (white light)• Distilled or deionized water

All experiment components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor admin-istered to or consumed by humans or animals.

Requirements

What is qPCR and How Does It Work?EDVO-Kit S-54

3




Background Information

THE POLYMERASE CHAIN REACTION (PCR)

In 1984, Dr. Kary Mullis revolutionized the fi eld of molecular biology when he devised a simple and elegant method to copy specifi c pieces of DNA. Recognizing that an initial step in DNA replication in a cell’s nucleus is the binding of RNA primers, Mullis discovered that he could replicate DNA in vitro using short, synthetic DNA primers and DNA poly-merase I. Furthermore, because researchers can specify a primer’s sequence to target a specifi c gene, this method allowed for the rapid amplifi cation of a selected DNA sequence in vitro. For the development of this technique, known today as the Polymerase Chain Reaction (or PCR), Mullis was awarded the Nobel Prize in Chemistry in 1993.

In order to amplify DNA, purifi ed double-stranded DNA is mixed with the short DNA primers, a thermostable DNA polymerase (Taq) and nucleo-tides. The mixture is heated to 94°C to “denature” (i.e., unzip into single strands by breaking hydrogen bonds) the DNA duplex. Next, the sam-ple is cooled to 45°C-60°C, allowing the primers to base pair with their target DNA sequences (a step known as “annealing”). Lastly, the temperature is raised again, to 72°C, the optimal tempera-ture at which Taq polymerase will extend the primer to syn-thesize a new strand of DNA. Each cycle (denaturation, annealing, extension) doubles the amount of target DNA (Figure 1). Today, a special-ized machine, called a “ther-mal cycler” or “PCR machine”, is used to rapidly heat and cool the samples. As a result, a PCR cycle can be completed in less than 5 minutes; 20-40 cycles produce suffi cient DNA for analysis.

3'5'

3'5'

5'3'

5'3'

5'

5'3'3'5'

5'3'

5'5'

Denature 94°C

5'

Extension72°C

3'5'

Separation of two DNA strands

=

Primer 1=

Primer 2=

5'3'5'

Anneal 2 primers 45°C

3'5'5'

5'5'

3'5'5'

5'

5'3'

5'

5'5'

5'3'

5' 3'

5' 3'

5'3'

5'3'

5'3'

5'

5' 3'

Cyc

le 1

Cyc

le 2

Cyc

le 3

Target Sequence

5'3'

5' 3'

5' 3'

Figure 1:The Polymerase Chain Reaction



4



PRINCIPLES OF QUANTITATIVE PCR

The products of conventional PCR are most often analyzed by agarose gel electrophore-sis. If a target DNA sequence is present in the starting material and is amplifi ed by the PCR reaction, a band of DNA will be visible when the gel is stained (Figure 2). Therefore, conventional PCR coupled with electrophoresis produces a “yes/no” qualitative result. In contrast, quantitative PCR (qPCR, also known as “real-time” PCR) can determine the exact amount of target DNA in the starting material by measuring the accumulation of DNA as the reaction progresses. Electrophoresis is not required because amplifi cation and quanti-tation of the DNA occur simultaneously.

Similarly to conventional PCR, each cycle of real-time PCR doubles the amount of the DNA in the sample (Figure 1). Mathematically, this doubling can be expressed as an exponen-tial relationship – if we begin with a starting copy number of m, then after n cycles, we will have m x 2n copies of our DNA target. For example, if we start with one copy of our target, we will have two copies after the fi rst PCR cycle, four after the second PCR cycle, eight after the third PCR cycle, and so on. After many cycles (regardless of the amount of DNA present in the starting material) the amount of DNA produced reaches a maximum where a product curve fl attens out, known as the plateau (Figure 3). This leveling off of the curve is due to the depletion of reaction components like primers and nucleotides and the loss of Taq polymerase activity.

In contrast to conventional PCR, real-time PCR samples contain special fl uorescent dyes that produce light when bound to double-stranded DNA (Figure 4). This allows the user to measure the amount of DNA in a sample as it is being synthesized. The amplifi cation is performed in a thermal cycler that can excite the fl uorescent molecules and detect the signal that they produce. A measured increase in fl uorescence directly relates to an in-crease in the amount of amplifi ed DNA in the sample. In early cycles of PCR, fl uorescence is low because there is not a lot of DNA present in the sample. As the number of cycles increases, the PCR product accumulates, and so fl uorescence increases. The cycle during which the fl uorescence reaches a set threshold is known as the quantifi cation cycle, or Cq (Figure 3). As the concentration of DNA template increases, the number of cycles it takes to reach the Cq decreases. For example, if the DNA template is present in the sample in low levels, it takes many cycles before the fl uorescence can be detected (high Cq). Conversely, if the target DNA is abundant in the starting material, the fl uorescence will increase to measurable levels relatively quickly (low Cq).

The exact number of target DNA molecules in a sample can be determined by comparing its Cq value to those from samples of known concentration using a standard curve. To create a standard curve, a DNA template is diluted over several orders of magnitude (for example, from microgram to picogram quantities), and the Cq is determined for each sample (Figure 5). Plotting Cq on the y-axis and the log10 of the known DNA con-centration on the x-axis results in a straight line. The equation of this line is used to determine the starting concentration of our unknown sample by substituting the measured Cq value into the equation.

Figure 2:Results of a conventional

PCR experiment as analyzed by agarose gel electrophoresis.

00

10 20 30 40

0.3

0.2

0.1

Exponentialphase

NonExponential

plateauphase

Flu

ore

scen

ce

Threshold line

Cq value

Figure 3:Graph showing the exponential

phase and plateau phase of PCR.

5




APPLICATIONS OF QPCR TECHNOLOGY

Real-time PCR is a commonly used technique in the both the research and the diagnostic laboratory because it is fast, sensitive, and requires less material and technical skill than traditional techniques like North-ern or Southern blotting. For example, microbiologists commonly use qPCR to both identify and quantify microorganisms in food and water samples. Physicians may use qPCR to establish the exact level, or titer, of a particular bacteria or virus present in a specifi c patient sample. Because qPCR can differentiate between specifi c strains of a particular pathogen (like infl uenza A and B), it is a powerful diagnostic and informational tool for health professionals.

qPCR can also be used to determine the extent that a specifi c gene is “turned on,” i.e., how much RNA is being transcribed from that particular gene. First, the RNA must be converted into DNA before it can be quantifi ed. This process, known as reverse transcription, creates a complementary DNA (cDNA) sequence from an RNA template. Once the cDNA is produced, qPCR can be used to quantify the amount of cDNA — and, by extension, the amount of original RNA — present in the sample. This is very useful when bio-technology companies need to determine the effects of experimental medications on specifi c biological pathways. For these reasons, qPCR has become an essential technique for today's scientists.

This experiment explores the principles of qPCR using colorful dye samples. Us-ing agarose gel electrophoresis, they will observe the relationship between cycle number and amount of DNA present within a sample. Students will perform data analysis to support these observations.

Figure 4

FF

F

F

TaqF

F F

F

F

Taq

F F F

A. Denaturation Step

B. Annealing Step

C. Extension Step

40353025201510

0 1 2 3 4 5 6 7

Log Starting Quantity, copy number

Qu

anti

fica

tio

n C

ycle

StandardsUnknowns

Correlation Coefficient: 0.999 Slope: -3.488 Intercept: 39.204 Y=-3.488 X=39.204

Figure 5


6



EXPERIMENT OBJECTIVE:

This experiment explores the principles of qPCR by analyzing colorful dye samples us-ing agarose gel electrophoresis. Students will observe the relationship between cycle number and amount of DNA present within a sample.

LABORATORY SAFETY

1. Gloves and goggles should be worn routinely as good laboratory practice.

2. Exercise extreme caution when working with equipment that is used in conjunc-tion with the heating and/or melting of reagents.

3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.

4. Exercise caution when using any electrical equipment in the laboratory.

5. Always wash hands thoroughly with soap and water after handling reagents or biological materials in the laboratory.

LABORATORY NOTEBOOKS:

Scientists document everything that happens during an experiment, including ex-perimental conditions, thoughts and observations while conducting the experiment, and, of course, any data collected. Today, you’ll be documenting your experiment in a laboratory notebook or on a separate worksheet.

Before starting the Experiment:

• Carefully read the introduction and the protocol. Use this information to form a hypothesis for this experiment.

• Predict the results of your experiment.

During the Experiment:

• Record your observations.

After the Experiment:

• Interpret the results – does your data support or contradict your hypothesis? • If you repeated this experiment, what would you change? Revise your hy-

pothesis to refl ect this change.

Experiment Overview

Wear gloves and safety goggles


7




Experiment Overview

After electrophoresis, transfer gel for visualization.

Analysis on white

light source

Attach safety cover,connect leads to power

source and conduct electrophoresis

Remove end blocks & comb, then submerge

gel under buffer in electrophoresis

chamber

Prepare agarose gel in

casting tray

5

4

2

1

( - )

Load eachdye sample inconsecutive wells

3

EDCBA

Gel pattern will vary depending upon the experiment.


8



60°C

1:001. 3.

4. 5.

7.

Caution! Flask will be HOT!

Concentratedbuffer

Distilledwater

Agarose

2.50x

Flask

60°C20min.

WAIT6.

Pour

Agarose Gel Electrophoresis

1. DILUTE concentrated (50X) buffer with distilled water to create 1X buffer (see Table A).2. MIX agarose powder with 1X buffer in a 250 ml fl ask (see Table A).3. DISSOLVE agarose powder by boiling the solution. MICROWAVE the solution on high

for 1 minute. Carefully REMOVE the fl ask from the microwave and MIX by swirling the fl ask. Continue to HEAT the solution in 15-second bursts until the agarose is completely dissolved (the solution should be clear like water).

4. COOL agarose to 60° C with careful swirling to promote even dissipation of heat.5. While agarose is cooling, SEAL the ends of the gel-casting tray with the rubber end

caps. PLACE the well template (comb) in the appropri-ate notch.

6. POUR the cooled agarose solution into the prepared gel-casting tray. The gel should thoroughly solidify within 20 minutes. The gel will stiffen and become less transparent as it solidifi es.

7. REMOVE end caps and comb. Take particular care when removing the comb to prevent damage to the wells.

IMPORTANT:

If you are unfamiliar with agarose gel prep and electrophoresis, detailed instructions and helpful resources are available at www.edvotek.com

ConcentratedBuffer (50x)

Size of GelCasting tray

7 x 7 cm

7 x 10 cm

7 x 14 cm

0.6 ml

1.0 ml

1.2 ml

+DistilledWater

29.4 ml

49.0 ml

58.8 ml

+TOTALVolume

30 ml

50 ml

60 ml

=

Individual 0.8% UltraSpec-Agarose™ GelTable

AAmt ofAgarose

0.23 g

0.39 g

0.46 g

Wear gloves and safety goggles


9




Agarose Gel ElectrophoresisReminder:

Before loading the samples, make sure the gel is properly oriented in the apparatus chamber.

8. PLACE gel (on the tray) into electrophoresis chamber. COVER the gel with 1X electrophoresis buffer (See Table B for recom-mended volumes). The gel should be completely submerged.

9. LOAD the entire sample (35-38 μL) into the well in consecu-tive order. The identity of each sample is provided in Table 1.

10. PLACE safety cover. CHECK that the gel is properly oriented. Remember, the DNA samples will migrate toward the positive (red) electrode.

11. CONNECT leads to the power source and PERFORM electro-phoresis (See Table C for time and voltage guidelines).

12. After electrophoresis is complete, REMOVE the gel and cast-ing tray from the electrophoresis chamber and VISUALIZE the results. No staining is necessary.

1X DilutedBuffer

8. 9.

10. 11. 12.( - )

( + )

1 2 3 4 5 6

Pour

Lane

1

2

3

4

5

Tube A Standard dyes with assigned bp equivalents

Tube B Sample after 10 cycles

Tube C Sample after 20 cycles

Tube D Sample after 30 cycles

Tube E Sample after 40 cycles

Table 1: Gel Loading

Time and Voltage Guidelines(0.8% Agarose Gel)

Min. / Max.Volts

150 125 75

15/20 min. 20/30 min. 35 / 45 min.

Table

CElectrophoresis Model

M6+ M12 & M36Min. / Max.

25 / 35 min. 35 / 45 min. 60 / 90 min.

50x Conc.Buffer

DistilledWater+

EDVOTEKModel #

Total Volume Required

1x Electrophoresis Buffer (Chamber Buffer)

M6+

M12

M36

300 ml

400 ml

1000 ml

Dilution

Table

B

6 ml

8 ml

20 ml

294 ml

392 ml

980 ml


10



Study Questions

1. List and describe the three basic steps of conventional PCR.

2. What are some differences between conventional PCR and real-time PCR?

3. What is the Cq? How is the Cq related to the initial DNA concentration?

4. Imagine that you are a physician and you are trying to treat bacterial infection in three differ-ent patients. You decide to use qPCR to determine whether or not the treatment is effective. A high bacterial load (over 10 pg of bacterial DNA) suggests that the infection is not being cleared and the treatment must change, whereas a low bacterial load (less than 10 pg of bac-terial DNA) suggests treatment is working.

Below is the data from qPCR test. Using the standard curve, determine the bacterial load of the three patients. Which patients are being treated effectively? Which patients need a differ-ent treatment?

Patient 1 – Cq = 25 Patient 2 – Cq = 18 Patient 3 – Cq = 32

0.0115

0.10 1.00 10.00 100.00 1000.00 10000.00

40

35

30

25

20

DNA (pg)

Qu

anti

fica

tio

n C

ycle


11




Instructor's Guide


12


INSTRUCTOR'S GUIDE What is qPCR and How Does It Work? EDVO-Kit S-54

OVERVIEW OF INSTRUCTOR’S PRELAB PREPARATION:

This section outlines the recommended prelab preparations and approximate time requirement to complete each prelab activity.

What to do: When: Time Required:

Prepare diluted TAE buffer

Prepare molten agarose and pour gel

40 min.

Prepare QuickStrips™

Up to one day before performingthe experiment.

Pre-Lab Preparations: Module I

FOR MODULE IEach Student Groupshould receive:• 50x concentrated buffer• Distilled Water • UltraSpec-Agarose™• Ready-to-Load™ Samples

NOTE:Accurate pipetting is critical for maximizing successful experi-ment results. EDVOTEK Series 100 experiments are designed for students who have had previous experience with micropipetting techniques and agarose gel electrophoresis.

If students are unfamiliar with using micropipets, we recom-mended performing Cat. #S-44, Micropipetting Basics or Cat. #S-43, DNA DuraGel™ prior to conducting this ad-vanced level experiment.

Carefully cut betweeneach set of tubes

EDV

OTE

K®

•

DO

NO

T BE

ND

A

B

C

D

E

F

G

H

CU

T H

ERE

A

B

C

D

E

F

G

H

CU

T H

ERE

A

B

C

D

E

F

G

H

CU

T H

ERE

CU

T H

ERE

A

B

C

D

E

F

G

H

CU

T H

ERE

A

B

C

D

E

F

G

H

A

B

C

D

E

F

G

H

SEPARATION OF PCR PRODUCTS BY AGAROSE GEL ELECTROPHORESIS

This experiment requires a 0.8% agarose gel per student group. You can choose whether to prepare the gels in advance or have the students prepare their own. Allow approximately 30-40 minutes for this procedure.

Individual Gel Preparation:

Each student group can be responsible for casting their own individual gel prior to conducting the experiment. See Module I in the Student’s Experi-mental Procedure. Students will need 50x concentrated buffer, distilled water and agarose powder.

Batch Gel Preparation:

To save time, a larger quantity of agarose solution can be prepared for shar-ing by the class. See Appendix B.

Preparing Gels in Advance:

Gels may be prepared ahead and stored for later use. Solidifi ed gels can be store under buffer in the refrigerator for up to 2 weeks.

Do not freeze gels at -20º C as freezing will destroy the gels.

Gels that have been removed from their trays for storage should be “an-chored” back to the tray with a few drops of molten agarose before being placed into the tray. This will prevent the gels from sliding around in the trays and the chambers.

SAMPLES FORMAT: PREPARING THE QUICKSTRIPS™

QuickStrip™ tubes consist of a microtiter block covered with a protective overlay. Each well contains pre-ali-quoted dyes.

Using sharp scissors, carefully divide the block of tubes into individual strips by cutting between the rows (see diagram at right). Take care not to damage the protec-tive overlay while separating the samples.

Each lab group will receive one set of tubes. Before loading the gel, remind students to tap the tubes to col-lect the sample at the bottom of the tube.

13



INSTRUCTOR'S GUIDEEDVO-Kit S-54 What is qPCR and How Does It Work?

Experiment Results and Analysis


14


INSTRUCTOR'S GUIDE What is qPCR and How Does It Work? EDVO-Kit S-54

S-54 Gel Idealized schematic: The relative positions of dye molecules are shown but are not depicted to scale.

Lane 1 A Standard dyes with assigned base pair equivalents B = Blue R = Red P = Purple Y = Yellow 2 B Sample after 10 cycles 3 C Sample after 20 cycles 4 D Sample after 30 cycles 5 E Sample after 40 cycles

3,500

1,500

800

450

1 2 3 4 5 6

YPR

B

In this qPCR simulation, the amount of dye increases as it proceeds through the qPCR reaction cycles, as shown in Lanes 2 through 5 in the above fi gures.

Please refer to the kit insert for the Answers to

Study Questions

A EDVOTEK® Troubleshooting Guide

B Bulk Preparation of Agarose Gels

Material Safety Data Sheets:

Now available for your convenient download on www.edvotek.com.

Appendices


16


APPENDICES What is qPCR and How Does It Work? EDVO-Kit S-54

Appendix AEDVOTEK® Troubleshooting Guides

PROBLEM: CAUSE: ANSWER:

Bands not visible on the gel

Ensure that the electrophoresis buffer was correctly diluted.

The electrophoresis buffer was not prepared properly.

Dye bands disappearwhen the gels are keptat 4° C.

Gel was not prepared properly. Make sure to prepare a 0.8% gel.

The dye molecules are small and will diffuse out of the gel.

The results must be analyzed upon the completion of electrophoresis

Very light colored bandseen after electrophoresis

Pipetting error.Make sure students pipet 35 µl of dye sample per well.

Poor separation of bands

Ensure that leads are attached in the correct orientation.

The dyes ran off of the gel because the polarity of the leads was reversed.

Contact the manufacturer of the electrophoresis unit or power source.

Malfunctioning electrophoresis unit or power source.

17



APPENDICESEDVO-Kit S-54 What is qPCR and How Does It Work?

Appendix B

To save time, the electrophoresis buffer and agarose gel solution can be prepared in larger quantities for sharing by the class. Unused diluted buffer can be used at a later time and solidifi ed agarose gel solution can be remelted.

Bulk Preparation of Agarose Gels

Bulk Electrophoresis Buffer

Quantity (bulk) preparation for 3 liters of 1x electro-phoresis buffer is outlined in Table D.

Batch Agarose Gels (0.8%)

For quantity (batch) preparation of 0.8% agarose gels, see Table E.

1. Use a 500 ml fl ask to prepare the diluted gel buffer

2. Pour 3.0 grams of UltraSpec-Agarose™ into the prepared buffer. Swirl to disperse clumps.

3. With a marking pen, indicate the level of solution volume on the outside of the fl ask.

4. Heat the agarose solution as outlined previously for individual gel preparation. The heating time will require adjustment due to the larger total volume of gel buffer solution.

5. Cool the agarose solution to 60°C with swirling to promote even dissipation of heat. If evaporation has occurred, add distilled water to bring the solu-tion up to the original volume as marked on the fl ask in step 3.

6. Dispense the required volume of cooled agarose solution for casting each gel. The volume required is dependent upon the size of the gel bed and DNA staining method which will be used. Refer to Appendix A or B for guidelines.

7. Allow the gel to completely solidify. It will become fi rm and cool to the touch after approxi-mately 20 minutes. Then proceed with preparing the gel for electrophoresis.

60˚C

Note: The UltraSpec-Agarose™ kit component is usually labeled with the amount it contains. Please read the label care-fully. If the amount of aga-rose is not specifi ed or if the bottle's plastic seal has been broken, weigh the agarose to ensure you are using the correct amount.

50x Conc.Buffer +

DistilledWater

Total Volume Required

60 ml 2,940 ml 3000 ml (3 L)

Bulk Preparation of Electrophoresis BufferTable

D

Batch Prep of 0.8% UltraSpec-Agarose™Table

EAmt ofAgarose

(g)

ConcentratedBuffer (50X)

(ml)+

DistilledWater(ml)

TotalVolume

(ml)+

3.0 7.5 382.5 390


18


APPENDICES What is qPCR and How Does It Work? EDVO-Kit S-54

what is qpcr and how does it work? - edvotekin 1984, dr. kary mullis revolutionized the ﬁ eld of...

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