Biol 2281, Spring 2016 E7: PCR Introduction, Procedure and Report

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Experiment 7: The Polymerase Chain Reaction (PCR) of human mtDNA

Objectives: At the end of the exercise, you should be able to 1. understand the concepts and principles of polymerase chain reaction. 2. understand and describe the major characteristics of mitochondrial DNA and single

nucleotide polymorphism of the control region sequences. 3. demonstrate laboratory techniques used in DNA isolation, PCR reaction and

visualization of PCR product by DNA agarose gel electrophoresis.

Introduction

1. Polymerase Chain Reaction - Xeroxing DNA

Copyright © 1994-2006 by Access Excellence @ the National Health Museum. Permission to use this material is granted for educational use. See website at http://www.accessexcellence.org/RC/AB/IE/PCR_Xeroxing_DNA.html.

Who would have thought a bacterium hanging out in a hot spring in Yellowstone National Park would spark a revolutionary new laboratory technique? The polymerase chain reaction (PCR), now widely used in research laboratories and doctor's offices, relies on the ability of DNA-copying enzymes to remain stable at high temperatures. No problem for Thermus aquaticus, the sultry bacterium from Yellowstone that now helps scientists produce millions of copies of a single DNA segment in a matter of hours. In nature, most organisms copy their DNA in the same way. The PCR mimics this process, only it does it in a test tube. When any cell divides, enzymes called polymerases make a copy of all the DNA in each chromosome. The first step in this process is to "unzip" the two DNA chains of the double helix. As the two strands separate, DNA polymerase makes a copy using each strand as a template. The four nucleotide bases, the building blocks of every piece of DNA, are represented by the letters A, C, G, and T, which stand for their chemical names: adenine, cytosine, guanine, and thymine. The A on one strand always pairs with the T on the other, whereas C always pairs with G. The two strands are said to be complementary to each other. To copy DNA, polymerase requires one other component: a primer. DNA polymerases, whether from humans, bacteria, or viruses, cannot copy a chain of DNA without a short sequence of nucleotides to "prime" the process, or get it started. So the cell has another enzyme called a primase that actually makes the first few nucleotides of the copy. This stretch of DNA is called a primer. Once the primer is made, the polymerase can take over making the rest of the new chain. A PCR vial contains all the necessary components for DNA duplication: a piece of DNA, large quantities of the four nucleotides, large quantities of the primer sequence, and DNA polymerase. The polymerase (in PCR) is the Taq polymerase, named for Thermus aquaticus, from which it was isolated. The three parts of the polymerase chain reaction are carried out in the same vial, but at different temperatures. The first part of the process separates the two DNA chains in the double helix. This is done simply by heating the vial to 90-95 degrees centigrade for 30 seconds. But the primers

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cannot bind to the DNA strands at such a high temperature, so the vial is cooled to 55-58 degrees C. At this temperature, the primers bind or "anneal" to the ends of the DNA strands. This takes about 30 seconds. The final step of the reaction is to make a complete copy of the templates. Since the Taq polymerase works best at around 75 degrees C (the temperature of the hot springs where the bacterium was discovered), the temperature of the vial is raised. The Taq polymerase begins adding nucleotides to the primer and eventually makes a complementary copy of the template. This completes one PCR cycle. The three steps in the polymerase chain reaction - the separation of the strands, annealing the primer to the template, and the synthesis of new strands - take less than two minutes. Each is carried out in the same vial. At the end of a cycle, each piece of DNA in the vial has been duplicated. But the cycle can be repeated 30 or more times. Each newly synthesized DNA piece can act as a new template, so after 30 cycles, 1 billion copies ( 230) of a single piece of DNA can be produced! PCR is valuable to researchers because it allows them to multiply unique regions of DNA so they can be detected in large genomes. Thanks to this procedure, one can make billions of copies of a single DNA molecule even though it is initially present in a mixture containing many different DNA molecules.

There are some limitations of PCR reactions: 1) Contamination of the DNA sample from other sources can cause problems. 2) Taq DNA polymerase has no proof-reading function, leading to a relatively high mutation rate (1/104); but now thermostable DNA polymerases with proof-reading activity are available.

2. Human Mitochondrial DNA

The following material was produced by the DNA Dolan Learning Center at Cold Spring Harbor Laboratory, Long Island, New York. See website: http://www.geneticorigins.org/geneticorigins/mito/mitoframeset.htm. Permission to use this material is granted for educational use.

Every human cell has a "second" genome, found in the cell's energy-generating organelle, the mitochondrion. In fact, each mitochondrion has several copies of its own genome, and there are several hundred to several thousand mitochondria per cell. This means that the mitochondrial (mt) genome is highly amplified. While each cell contains only two copies of a given nuclear gene (one on each of the paired chromosomes), there are thousands of copies of a given mt gene per cell. Because of this high copy number, it is possible to obtain a mt DNA type from the equivalent of a single cell's worth of mt DNA. Thus, mt DNA is the genetic system of choice in cases where tissue samples are very old, very small, or badly degraded by heat and humidity. Under good circumstances - working from fresh cell samples - mt DNA is the easiest human DNA to amplify by PCR. This experiment examines a 440-nucleotide sequence from the noncoding region of mt genome. Hand cycling is a realistic alternative to automated thermal cyclers, and the high yield of amplified product can be visualized in an agarose gel with a variety of stains. Because each student is amplifying the same region, the gel electrophoresis results will also be the same for each. However, amplified student samples may be submitted to our Sequencing Service, which will generate student mt DNA sequences and post the results on our Sequence Server. Comparison of control region sequences reveals that most people have a unique pattern

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of single nucleotide polymorphisms (SNPs). These sequence differences, in turn, are the basis for far-ranging investigations on human DNA diversity and the evolution of hominids.

The entire DNA sequence of the human mt genome - 16,569 nucleotides - was determined in 1981, well in advance of the Human Genome Project. The mt genome contains 37 genes, all of which are involved in the production of energy and its storage in ATP. Thirteen of these genes encode proteins involved in oxidative phosphorylation. The remaining genes encode tranfer RNAs (22 genes) and ribosomal RNAs (2 genes) that translate the proteins' genes within the mitochrondrion. Mammalian mt genes use a slightly different genetic code than nuclear genes, where UGA = tryptophan, AUA = methionine, and AGA and AGG = stop.

Genes take up the majority of the mt genome. However, a noncoding region of approximately 1,200 nucleotides spans both sides of the arbitrary "0" position of the mt genome and goes by three confusing terms: control region, D-loop, and hypervariable region. Control region refers to the fact that this region contains the signals that control RNA and DNA synthesis. A single promoter on each DNA strand initiates transcription in each direction, and a single origin initiates replication of each strand. D-loop refers to the early phase of replication, when the first newly-synthesized strand displaces one of the parental strands, forming a "bubble" or loop. The DNA sequence of the control region is termed hypervariable, because it accumulates point mutations at approximately 10 times the rate of nuclear DNA.

3. The puReTag Ready-To-Go PCR Beads From “GEHEALTHCARE”.

Each bead is a premixed, predispensed reaction for PCR featuring PuReTaq DNA polymerase, when suspended in a final volume of 25 μl, will contain the following:

• 2.5 units of puReTaq DNA Polymerase • 10 mM Tris-HCl, pH 9.0 at room temperature

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• 50 mM KCl • 1.5 mM MgCl2 • 200 μM of each dNTPs

Day 1 Material: per student

Marker pen 0.9% NaCl solution, 10 ml in a paper cup Three of 1.5 ml tubes and tube rack 10% Chelex®, 100 µl ( return colored tube after use) One dropper pipet for transferring the mouse rinse to the 1.5 ml tube micropipet (P200 and red-5 µl) One yellow tip box Primer/loading buffer mix (20 µl): provided by the TA Ready-to-Go PCR Bead (0.2 ml tube): provided by the TA

Shared items

Microcentrifuge Hot plate for boiling water Thermal cycler

The following procedure was based on information produced by the DNA Dolan Learning Center at Cold Spring Harbor Laboratory, Long Island, New York. Parts of the procedure were modified to suit the experimental equipment and supplies available in the UG lab at UT Dallas. Permission to use this material is granted for educational use. Part I: Total DNA Isolation by Saline Mouthwash

1. Label the cap of your three microcentrifuge tubes with initial and 1, 2 and 3, respectively.

2. Obtain a colored microtube (Chelex) from common bench. Tap the colored tube or use votexerto mix and resuspend the white particles throughly. Before the white particles settle down, quickly pipet 100 µl of Chelex into a clean 1.5 ml tube (Tube 1). Set tube 1 aside.

3. Return the colored tube to common bench right away. 4. Pour 10 ml of the saline solution into mouth and vigorously swish for 30 seconds. 5. Expel saline solution into a paper cup. 6. Swirl to mix cells in the cup.

7. Use dropper pipet to transfer 1.5 ml of the liquid to a 1.5 ml tube. (Tube 2)

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8. Place tube 2, together with other samples, in a balanced configuration and spin in a microcentrifuge for 1 minute.

9. Carefully pour off supernatant (liquid layer) into sink. Be careful not to disturb the cell pellet at the bottom of the tube. A small amount ( 0.1 ml) of saline should remain in the tube.

10. To collect more cells in tube 2, repeat step 7 to step 9 one more time using tube 2.

11. Resuspend and mix cells (pellet) in remaining saline ( 0.1 ml) by pipetting up and down with the P200 micropipet.

12. Withdraw 40 µl of cell suspension from Tube 2, and add to the tube containing 100 µl of Chelex (Tube 1). Shake well to mix.

13. Close the cap. Boil cell sample in tube 1 for 10 minutes in a boiling water bath. (After the water comes to a boil, reset the hot plate temperature setting to between 6 and 7).

You may obtain PCR Bead in a 0.2 ml tube (reaction tube) and 20 µl of the primer/loading buffer mix from TA during the 10 minutes. (See step 1 in Part II).

14. After boiling, place tube 1 in a balanced configuration with other sample tubes, spin for 1 minute.

15. Transfer 20 µl of supernatant (top liquid layer containing the DNA) to a clean 1.5 ml tube (Tube 3). Avoid cell debris and Chelex beads.

Note:

1) From Bio-rad: “Chelex 100 molecular biology grade resin is a highly pure, pipettable, nuclease and ligase inhibitor-free chelating resin specifically designed and certified for extraction of PCR-ready template DNA. Chelex 100 molecular biology grade resin accommodates the stringency required for a PCR-quality product by ensuring the complete removal of PCR inhibitors (contaminating metal ions that catalyze the digestion of DNA).”

2) Boiling the cell mixture serves to lyse the cell to release the DNA and at the same time, inactivate cellular proteins that may interfere with the PCR reaction.

Part II: DNA Amplification by PCR

1. Obtain a PCR reaction tube (containing a small Ready-To-Go PCR Bead) from TA.

( TA station) Use a micropipet with a fresh tip to add 20 µl of the appropriate primer/loading buffer mix to small PCR reaction tube. Tap the tube to dissolve bead. The Ready-To-Go PCR Bead is a patented product that contains the Taq polymerase.

2. Use red fixed-volume micropipette and fresh tip to add 5.0 µl of human DNA (Tube 3 from Part I) to small PCR reaction tube, and tap to mix.

Label the cap of small PCR reaction tube with your sign-in number and section number. The volume of your sample in PCR tube should be ______ µl now.

3. Place your small PCR reaction tube on the ice bucket next to the PCR machine.

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Cleaning Up: 1. Dispose the tips, microcentrifuge tubes, droppers and paper cups in regular trashcan. 2. Place tip box, tube rack and micropipette to original containers.

Store your small PCR reaction tube on ice until ready to amplify according to the following profile. Program thermal cycler for 30 cycles according to the following cycle profiles. At the end of the program, the amplified DNAs will be held at 4°C. Your samples will be transferred to a freezer until next lab to be analyzed by gel electrophoresis.

Step 1: Denaturation: 30 sec at 940C

The reaction mix is heated to about 940C. At this temperature, the dsDNA dissociate into single strands

Step 2: Annealing of Primers: 30 sec at 580C

Next, the solution is allowed to cool to about 580C. As it cools, the single strands of DNA re-associate into double strands according to sequence complementarity. However, because of the large excess of primer, each strand of the DNA template base-pairs with a complimentary primer, leaving the rest of the DNA template single stranded.

Step 3: Primer Extension: 30 sec at 720C

Using the primers, the heat-stable DNA polymerase (Taq polymerase) copies the rest of the DNA template as if it were replicating DNA. When it is done, the primer has been lengthened into a complimentary copy of the entire single-stranded fragment. Because both DNA template strands are replicated, there are now two copies of the original fragment.

Step 4: Repeat the above three steps 30 times

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DAY 2 Part III: DNA Analysis by Gel Electrophoresis

1. 0.5X TBE buffer 2. 1µg/ml ethidium bromide (provided by the TA) 3. 100 bp DNA molecular size marker 4. agarose 5. micropipet (P20) and yellow tip box 6. electrophoresis equipment and power supply

Procedure

1. (TA) Prepare a 1.5% agarose gel with a total volume of 50 ml in 0.5X TBE. Heat it to melt the agarose; How much agarose to weigh? _________________ grams.

2. TA will add 1 µl of EB solution (10mg/ml) to your melted agarose gel BEFORE pouring to the gel tray.

3. Use a micropipet with a fresh tip to load 10µl PCR product into your assigned well of the gel. Expel any air from the tip before loading, and be careful not to push the tip of the pipet through the bottom of the sample well.

4. (TA) Load 5 µl of the 100 bp-DNA size marker (ladder) into one lane of the gel. 5. Electrophorese at 130 volts for 35-40 minutes. Adequate separation will have occurred

when the cresol red dye front has moved at least 50 mm from the wells. 6. Check the map on page 3, based on the locations of the two primers to predict the size

of the PCR product. The size is predicted to be ___________bp. Examine the gel using UV lamp, estimate the size of your PCR product by comparing to the 100 bp-DNA size marker (ladder).

Part IV: DNA Sequencing by Dideoxy Chain Termination

Your amplified mt DNA samples will be sent to the DNA Dolan Learning Center for sequencing. Animation of cycle sequencing can be viewed at http://www.geneticorigins.org/mito/media.html. After four to six weeks, you can access sequences by using a tool "Sequence Server." at http://www.bioserver.org/sequences/. To use this tool, you will need access to the internet and either Netscape 4.0 (or better) or Internet Explorer 4.0 (or better). After logging in (click “ENTER”) to Sequence Server, you should click the "Manage Groups" button at the top of the screen. This will open the Manage Groups window. In this window, choose "Classes" from the popup menu “Sequence Sources” on the upper-right. A new screen will appear, and you will be able to see your class's name in the list presented. To select your class, click on the checkbox next to your class, and click the OK button. This will put your class on your worksheet. To compare sequences, you need to have more than one sequence on the worksheet. To add a sequence, select the desired sequence from the popup menu at the bottom of your class. Do this for as many sequences as you want. Then, click the checkbox for each sequence you wish to include in a comparison, then click on the "Compare" button. Sequence Server will open a new window showing the results of your analysis. You can include the sequence of Neandertal mtDNA by choosing prehistoric DNA from “sequence sources”.

Biol 2281, Spring 2016 E7: PCR Introduction, Procedure and Report

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Post Lab Report (20 pts total): Include title page and questions in your report (1pt)

1. Presentation of the picture of the gel, with proper labeling of each lane, wells, the sizes of the marker bands, and the anode and cathode. ( 2 pts) (photos posted on eLearning)

2. How did you predict the size of the PCR product ( Human mitochondrial genome map on page 3)? What is the estimated size of your PCR product based on the DNA size marker loaded on your gel when you compare it to the ladder? Why was the 100 base-pair ladder used instead of 500 bp ladder for this exercise? (3 points)

3. Each round of PCR doubles the number of DNA molecules present in the reaction. How many molecules can be generated from one of your DNA template molecules after 30 cycles? ( 1 points)

4. You amplified a segment of your own mitochondrial DNA.Do you expect the sequences of your PCR product vary from those of your classmates? Why or Why not? ( 2 points)

5. What does the term “denaturation” mean in relation to DNA? ( 2 pts) 6. What does the term “annealing” mean in relation to DNA? What kinds of chemical bonds

are formed when two strands of DNA anneal? ( 2 points) 7. The process of PCR is dependent upon the use of certain DNA polymerases.

a) Why are they special and where do they come from? ( 2 points) b) Why does the PCR reaction catalyzed by the Taq DNA polymerase have a

higher mutation rate than the normal DNA replication process found in living cells? (1pt)

8. What is the basis of separation of DNA molecules in the agarose gel that you run in this experiment? What are the electrical charges of the DNA molecules? (2 pts)

9. What reagent is added to the 0.5X TBE buffer to allow visualization of the DNA bands in the agarose gel? Describe how you can see the DNA after the gel running is stopped. (2 pts)

BIOL2281 E7

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Experiment 7: Extracting and Amplifying mtDNA

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1. Polymerase Chain Reaction (PCR)

2. Mitochondrial DNA

3. Experiment 7 Content

The Purpose of PCR

• Within a few hours, greatly amplify unique regions of DNA so they can be detected in large genome

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• This amplified sample then allows for size determination, restriction mapping and nucleotide sequencing– Basic Research

– Applied Research• DNA “fingerprinting”--forensic science

• Paternity determination

• Disease diagnosis

1. Target DNA (purified or a crude extract)

2. A pair of primers specific for the target DNA

The reaction mixture

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DNA

3. Free deoxynucleotides (dATP, dGTP, dCTP, dTTP): building blocks/energy source

4. Heat stable DNA polymerase (Thermus

aquaticus)

5. Buffer (containing cofactor, Mg++)

The basic protocol—The Reaction Mix

Target DNA5’ 3’

3’ 5’

4

primersA

B Freenucleotides

Taq DNApolymerase

Mg2+Mg2+

Mg2+

Mg2+

Mg2+

Mg2+

Buffercontainingmagnesium

A pair: two primers

Usually about 20 nucleotides in length

Designed to flank the region to be amplified

Must be complementary to the 3’ end of the

Primers

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5’ 3’

3’ 5’

5’5’ A

B

gene to be amplified

Quantity: In large excess

1. Step 1:Denaturation of DNA

2. Step 2: Annealing of primers to DNA

The basic protocol

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3. Step 3: Extension by polymerase

4. Repeat 30-35 times

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Step (1)--denaturation

Target DNA5’ 3’

3’ 5’

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95oC

5’ 3’

3’ 5’

Step (2)--annealing5’ 3’

3’ 5’

Denatured Target DNA

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~55oC

5’ 3’

3’ 5’

5’5’ A

B

primers

A

B

Step (3)--extension

72oC

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3’ 5’

5’ 3’

Taq polymerase

One One billion in about 2 hours!

• At the end of each cycle, the amount of DNA has doubled

Media links• http://www.dnalc.org/resources/animations/

index.html• http://www.sumanasinc.com/webcontent/an

imations/content/pcr html

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imations/content/pcr.html• http://www.bioservers.org/bioserver/index1.

html• http://www.geneticorigins.org/mito/media.h

tml• http://www.youtube.com/DNALearningCen

ter#p/f/15/JRAA4C2OPwg

What makes it work?

• DNA thermal cycler

• Taq DNA polymerase!– Taq was isolated from Thermus aquaticus a

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Taq was isolated from Thermus aquaticus, a bacteria that grows in hot springs (~75oC)

– This organism’s enzymes have adapted to the high temperature, so they can survive cycling through the high temperatures;

– What is the optimal temperature of Taq DNA plymerase? _____________

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The PCR machine

• Very rapidly changes of the temperature between the various

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stages of the PCR process

• Programmable for use with many different cycling parameters

Mitochondrial DNA Template

• The mtDNA Genome • Comparing mtDNA with nuclear DNA• The Expected PCR Product

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The Expected PCR Product– Where are the two primers?– What is the expected size of the PCR

product?– Sequence variations: single nucleotide

polymorphism (SNPs)

5'-TTAACTCCACCATTAGCACC-3'5'-GAGGATGGTGGTCAAGGGAC-3'

Human Nuclear DNA Human mtDNA

Very Large: About 3 billion (3,000,000,000) base pairs of DNA.

Very Small: Exactly 16,569 base pairs of DNA.

Very Complex: Encodes about 23,000 proteins.

These proteins undertake a vast f k

Comparatively simple: 37 Genes of which 13 code for

proteins, others code for tRNA or RNArange of tasks. rRNA

Very Long Chains-Linear Small Circular Loop

Two copies of a given nuclear gene per cell:

One maternal and one paternal.

Many copies per cell: Each cell has many mitochondria,

and each mitochondrion has many copies of mtDNA.-are inherited in a maternal lineage.

Single Nucleotide Polymorphism (SNP)

• a small genetic change, or variation, that can occur within a person's DNA sequence.– AAGGTTA

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– ATGGTTA

• SNPs are stably inherited in a Mendelian fashion-used to study human diversity and evolution

Experiment 7 Content:

• Part 1: Total DNA isolation

• Part 2: mtDNA amplification by PCR

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• Part 3: PCR product analysis by gel electrophoresis

BIOL2281 E7

Dr. Wenju Lin, Spring 2016 4

The PCR Reaction in Our Experiment:1. 20 l Primer/Loading Buffer mix

– Two primers– Cresol Red Loading dye– Sucrose

2 5 l of Human Total DNA

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2. 5 l of Human Total DNA3. puReTaq Ready-To-Go PCR Bead; when

reconstituted in a final volume of 25 l – 2.5 units of puRe Taq DNA polymerase– 10 mM Tris-HCl, pH 9.0 at Room Temp.– 50 mM KCl– 1.5 mM MgCl2

– 200 µM of each dNTPs

The PCR program in our experiment:

• Step 1: Denaturation at 940C for 30 sec

• Step 2: Annealing at 580C for 30 sec

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• Step 2: Annealing at 580C for 30 sec

• Step 3: Primer Extension at 720C for 30 sec

• Step 4: Repeat the three steps for 30 cycles

Sample PCR Result

• The loading of the gel;

• The DNA Size marker;

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marker;

• The PCR band;

• The primer-dimer;

DNAs migrate to the (+) electrode (anode)

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What factors influence the rate of migration in DNA agarose gel?

• Molecular size of the DNA– The mobility of linear DNA fragments is inversely

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– The mobility of linear DNA fragments is inversely proportional to the log10 of their molecular weight.

• The conformation of DNA• The agarose concentration• The buffer • The applied voltage

Effect of DNA Sizes on Mobility

The mobility of linear DNA fragmentsis inversely proportional to the log10 of their molecular weight.

Unit: Base pairs

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Effect of DNA Conformation on Mobility

• Circular form vs linear DNA

• Supercoiled vs nicked circles;

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Visualizing DNA by Ethidium Bromide

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1 g/ml of EB included in the agarose gel or TBE buffer

Ethidium Bromide

• Intercalates into the DNA molecules

• Fluorescence– UV radiation at 254 nm is absorbed by the DNA and

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transmitted to the bound dye

– The energy is re-emitted at 590 nm in the red-orange region of the the spectrum

– Wear goggles to protect from UV

• Carcinogen– Wear gloves

• Agarose concentration- 1.5%

• Buffers: 0.5X TBE – 4.5 mM Tris; 4.5 mM Boric Acid; 0.1 mM

EDTA; pH 8 3

In Our Experiment:

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EDTA; pH 8.3

• Voltage: 120-130 V

• M.W. Marker– 100 bp ladder

• Chelex : ( From Bio-rad) complete removal of PCR inhibitors such as contaminating metal ions that catalyze the digestion of DNA

Quiz Review1. What is the direction of synthesis of the new DNA strand in each cycle?

From 5’ to 3’ or from 3’ to 5’?2. What determines the size of the final PCR product?3. What temperature is used to denature the DNA template? 4. What temperature is used to synthesize the new DNA strand?5. What is unique about the Taq DNA polymerase?6. Do you need to have a pure template DNA sample (free of other DNA

sequences) to allow specific amplification of a segment of the template DNA?

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q ) p p g p7. What would be the consequence if the reaction contained proteases

(enzymes that can degrade proteins into amino acids)?8. What would be the consequence if the reaction contained DNases

(enzymes that can degrade DNA into nucleotides)?9. What two components need to be added to the puReTaq Ready-To-Go

PCR Beads to set up a PCR reaction?10. (True or False) The amplification of the original target DNA is exponential.