Copying DNAPolymerase Chain Reaction

Polymerase Chain ReactionMost widely used amplification techniqueEnables researchers to produce millions

of copies of a specific DNA sequence in approximately two 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.

PCR can be used to amplify DNA that is 40,000 years old. It has been used to amplify DNA from mummies and woolly mammoth

Tools & Tasks

We are going to actually copy (replicate) DNA in a laboratory setting. To do this we will need to learn how some lab equipment works as well as how we work the lab equipment.

You will be asked to perform some tasks successfully as well as know why you are performing that certain task.

This world of DNA technology is fascinating and EXPENSIVE. Because the chemicals used are so expensive, the industry has learned to use very small amounts.

A microliter is the industry standard.

A microliter is one millionth of a liter and is represented by the Greek symbol “μL”It is 1/1,000,000 of a liter or 0.02% of a drop.

A special tool is used to measure out and dispense microliter. It is called a micropipette.

The act of using the micropipette to dispense a liquid is called aliquotting.

A. These are the parts of a micropipette a. Plunger button b. Tip ejector button c. Volume adjustment dial/Indicator d. e. Shaft f. Attachment point for a disposable tip

The micropipetors in this laboratory come in three different sizes each of which measures a different range of volumes. The three sizes are P20, P200 and P1000. These sizes are noted on the top of the plunger button. Size Micropipette Range of volumes measured P20 0.5-20μl P200 20-200μl P1000 100-1000μl

Adjusting Volume on micropipettes

Volume Adjustment Dial: The black volume adjustment dial near the top of the micropipette allows you to adjust the volume that is measured. It can be dialed to the left or right to increase or decrease the volume.

The digital readout shows the volume that will be measured. As you turn the volume adjustment dial, the numbers in the digital readout will change.

Micropipetting Step 1: Set the dial to the desired amount. Liquids are never drawn directly into the

shaft of the pipette. Instead, disposable plastic tips are attached to the shaft. There are 3 sizes of tips. The larger blue tips are used for the P1000. The smaller yellow tips are used for the P20 and clear tips for the P200.

The tips are racked in plastic boxes with covers. When you receive a box, it will be sterile. Please be careful when touching box or tips not to contaminate them. The box should be closed when not in use to prevent air-born contamination.

Step 2: Insert the Tip Select the correct size tips. Open the box without touching the

tips with your hands. Hold the bottom of box stable with free hand.

Insert the micropipette shaft into the tip and press down firmly. This will attach the tip to the shaft.

Remove the micropipette with the tip attached.

Close the box without touching the tips with your hands.

Step 3: Depress the plunger to the first position.The micropipette has a plunger that dispenses the liquid. The plunger has two important positions. When depressed you will feel “resistance” at the first position. This position is used to pick up liquid. Step 4: Insert the instrument into the liquid to be dispensed. Only the tip should come in contact with the liquid, never the pipette itself.

Step 5: Gently release the plunger.This accurately picks up the desired amount of liquid.Direct the tip of the plunger to the container receiving the liquid.

Step 6: With pressure you can push the plunger past the first position to the second. This one is used to expel the liquid. This will deliver the aliquot or exact amount of liquid desired.

Perform the procedure over a countertop for the safety of the instrument.

REVIEW

1.What does PCR stand for?

2.What is the symbol for a microliter?

3.How much of a liter is a microliter?

4.How much of a drop is a microliter?

5.How many sizes of micropipette are there?

6.What is meant by the term “aliquot”?

7.Describe the steps to aliquot a liquid.

8.Label the parts of the micropipette below

The instructor will now demonstrate the use of a 1000uL pipettor.

You are then to aliquot 1000uL of milk to a plastic reaction tube.

Once completed, write your initials on the side of the tube and compare the amount of milk to the sample the instructor is holding.

If you are off, repeat the process until your tube contains the desired amount.

Microcentrifuge• Scientists have found that there is

an inexpensive method to separate liquid mixtures according to their molecular weights.

• You simply put a mixture into a plastic reaction tube and spin it at a high rate of speed for a few minutes.

• Centrifugal force will separate out each member of the mixture according to its molecular weight.

• The heaviest component goes to the bottom of the tube.

Once you have aliquotted 1000 uL of milk into a reaction tube, insert that labeled reaction tube, hinge up, into the round drum of the microcentrifuge.

Spin the sample for 2 minutes.

Remove the sample and see if you can count how many of layers are present.

Using prior knowledge of milk, what do you think the layers are made of?

See if you can remove the top layer with the micropipette and a new tip.

Review1. What is the machine that spinned your

milk so it would separate into layers?2. What determines the layers?3. What is the tube called that is spinned?4. What is that outward force used in

separating during spinning?5. Heaviest or lightest component on top?6. Was the cream heaviest or lightest in

your milk?

Vortexing a Sample Vortexing is a simple word for mixing.

Tapping the reaction tube on the table does a superb job of mixing the chemicals inside the tube.

You can’t tap too hard however, the tube will break.

Sometimes liquid components get stuck in the top of the tube. In this case you would use a “snap vortex” to force all liquid into the bottom of the tube.

You simply hold the tube firmly between your fingers and snap your wrist once or twice. The abrupt movement forces the liquid to the bottom of the tube.

PCR Polymerase Chain Reaction

A technique which is used to copy a specific region of DNA.

The copy of the original DNA molecule is copied and in turn the copy of the copy is copied.

This allows us to create enough DNA to be adequately tested.

This technique can be used to identify with a very high-probability, disease-causing viruses and/or bacteria, a deceased person, or a criminal suspect.

Since most human DNA (95%) is identical, it is not beneficial to copy DNA just anywhere.

We need to copy the areas that are different.

Because of this we need a system that lets us choose the area of DNA to be copied.

PCR Polymerase Chain ReactionThree steps…

◦Denaturation – using high temperature unwind and unzip DNA

◦Annealing – Complementary DNA strand is hybridization using DNA primers

◦Extension – DNA strand synthesis using DNA polymerase.

More explanation…

Step 1 – Denaturing To Copy DNA we need to:

a) unwind DNAb) unzip DNA

In a regular cell this requires an enzyme Helicase (aka Protein Helper).

Scientists found that the easiest way to unwind and unzip DNA is to heat it which is called “denaturing”. This is done at about 90oC.

We also need an enzyme to glue the side chains of the nucleotides together once the A’s have bonded to the T’s and the C’s have bonded to the G’s. This enzyme is called a DNA Polymerase.

PROBLEM…When they heated DNA in the lab to denature it, they found the DNA Polymerase enzyme quit working. This was a big problem.

SOLUTION…They then found some organisms living in the boiling waters of Yellowstone Park and discovered that these creatures had a DNA polymerase that liked heat. Problem solved – use the heat resistant DNA polymerase to glue the side chains together.

In our lab we will use human DNA and a special heat resistant DNA polymerase. This will all happen in a plastic reaction tube.

Scientists still kept running into one big problem. The heat resistant DNA polymerase still did not work. Further study showed that it needed a starting point to begin copying DNA.

Step 2 – Annealing Scientists then created a fake piece of DNA

to become the starting point. This fake DNA is called the “Primer”.

Primer is A specially designed, synthetic

DNA, that allows a DNA polymerase to attach to the target DNA and begin copying.

In other words – it provides a starting point, that we pick, to start the DNA copying process.

Two primers are needed to amplify a particular region of target DNA.

Primers are man made so they can be engineered to attach to any point in any DNA.

They can be purchased on-line so they are easy to get.

They are expensive – this we measure them in microliters.

We also need the raw materials to make DNA, (the nucleotides). We buy a liquid containing millions of As, Cs, Gs, and Ts. This liquid is called the “Master Mix”.

We will put it in the reaction tube with the primers and target DNA.

Step 3 ExtensionDNA Polymerase enzyme synthesizes

new complimentary strands of DNA by the extension of primers - assemble and glue the new nucleotides into place

Remember…there are 3 steps involved in copying DNA in PCR:1. Denature the Molecule - unwind and

unzip the DNA (break the hydrogen bonds)

2. Primers Annealing – primer attached to the DNA right next to the target DNA.

3. Extension - DNA Polymerase enzyme synthesizes new complimentary strands of DNA by the extension of primers - assemble and glue the new nucleotides into place

Quiz1. PCR stands for what?2. Is PCR capable of replicating a single molecule of

DNA into millions of copies?3. Put the following three steps of PCR in order: Primer

annealing, Polymerase extension, Denaturing4. Explain what each of the following terms and their

functions: microcentrifuging vortexing Helicase DNA Polymerase Primer Master mix

5. There was a problem with the second step of PCR that was solved with a discovery at a national park. Explain this problem and the discovery.

Movie Quiz

What do we do once we have DNA?

We Need a process that will show us how every person’s DNA is different.

We will use a process called

“Gel Electrophoresis”.

Gel Electrophoresis Steps1. DNA is put into a gel called

“acrylamide”.2. DNA fragments are separated

with electricity.3. DNA is then stained so it is

visible and can be analyzed.

Here are two polyacrylamide gels containing

DNA.The bands are actual DNA.

The gel electrophoresis setup has a tank, gel tray, gel and power supply.

The whole procedure happens in the tank which is filled with a buffer.

Buffers are added. Buffers are chemicals that provide the optimal environment for chemical reactions to happen….

You will load the gel with your DNA as it sits in the tank. This is not as easy as it sounds because the tank is filled with a buffer called TAE and the gel is submerged in the TAE. You will do this with the micropipettor.

You will be aliquotting to a target that is underwater. (Don’t worry, it isn’t that hard. )The DNA is dense so it will sink into the well.

You will do this with the micropipettor. ◦You will add 15uL of PCRd DNA to a

small well in the gel.

Once loaded, we will turn the machine on and it will pass an electrical current through the gel.

DNA has a negative charge and the electricity is negative so the DNA will migrate down the gel toward the positive electrode.

Small pieces of DNA travel a great distance down the gel, large pieces, not so much.

This is a double Gel containing DNA . You can see the wells on the left side of the DNA run. To start the process, DNA is aliquotted into these wells.

Upon addition of electricity, DNA will

move away from the

wells. This band of DNA is larger so it didn’t travel as far as its neighbor.

This Band of DNA is smaller

so it went farther down

the gel.

Once we have passed an electrical current through the DNA, we will stain the molecule to see where it went.

The DNA will form bands like shown below. The band is actual DNA and its position in the gel is

crucial to our understanding.

Analyzing ResultsThe question is; “How will we be able to tell

one person’s DNA from another?”

We are going to look at an area of DNA that is polymorphic. Has lots of different DNA patterns because of our individual add-ins called “insertions”.

We are going to copy between two targets in the DNA yet the amount of DNA between these two targets is different for every human being, (because the area in-between the start points is polymorphic).

One person may have 440 base pairs while another may have 660, and yet another may contain 1100.

We will add a molecular ruler called an “Allele Ladder” to the gel so we can see how many base pairs are in each person’s DNA. The ruler contains DNA of all sizes so we can compare lab DNA and know approximately how many base pairs are in each band of DNA.

This is the molecular ruler. It identifies the size of DNA Bands by

comparing their positions in the gel.

Analysis “Fast Stain” is used. It is positive

charged so it “sticks to the DNA pieces.

Once we have the gel stained with “Fast Stain” the DNA is visible and we can draw conclusions.

If you have crime scene DNA and compare it with suspects, the suspect possesses bands of DNA that directly match up with the crime scene DNA is the perpetrator, or as the police say “Perp”.

Review….1. PCR stands for what?2. What is the symbol for microliter?3. Microliter is how much of a liter? How

much of a drop?4. What is the tool used to measure

microliter?5. What is the act of using this tool called?6. Draw this tool and label the plunger

button, tip ejector button, volume adjustment dial, shaft, and disposable tip.

7. Describe the six steps in using this tool.

Review…

1. What is the function of microcentrifuge?

2. How are substances separated in microcentrifuging?

3. What force is used in microcentrifuging?

4. Vortexing the sample does what?

5. Describe two vortexing methods.

Review…1. Explain the function of the enzyme

Helicase (Protein Helper)2. Explain the function of the enzyme

Polymerase?3. What does Yellowstone have to do with

Polymerase enzyme?4. What is the purpose of the Primer?5. How many primers are needed to amplify

a particular region of target DNA?6. Are primers man made (synthetic) or

natural; expensive or cheap?

Review….

1. What kinds of materials obtained from a crime scene might contain DNA?

2. Why do you need to perform PCR on DNA obtained from a Crime Scene.

3. What might you see if you ran a DNA sample extracted evidence on a gel before PCR?

4. What is a genotype?5. What is allele?

Review…What is in Master Mix?What are the three steps in PCR cycle?Explain each step.Why does DNA move through the

agarose gel?What property does electrophoresis

separate the bands by?What is an allele ladder?How is the DNA visualized on the gel?