Lab 6: Polymerase Chain Reaction

Question 1

What is PCR?

Answer 1

A method used to amplify a specific fragment of DNA from a complex mixture of DNA. (amplify a target gene or segment over 1 billion times without affecting any other fragments of DNA)

Question 2

What is DNA barcoding?

Answer 2

Sequencing a short stretch of DNA. The DNA provides a unique way to identify the species, much like UPC barcodes on products in stores that are used to uniquely identify the product.

Question 3

Typical PCR reaction

Answer 3

1. 95 degrees celsius- denaturation
2. 50 degrees celsius- annealing
3. 72 degrees celsius- synthesis

Question 4

Why do we use Taq polymerase in PCR?

Answer 4

Enzymes have optimal temperatures and many enzymes would degrade at high temperatures. Taq polymerase is an enzyme that has been purified from Thermus aquatics, a bacterium that grows in hot springs. This bacterium has evolved over millions of years to grow in extremely high temperatures, so the enzyme will not degrade at 95 degrees celsius. In fact, the 72 degrees celsius temperature for extension is the optimal temperature for Taq polymerase to synthesize DNA.

Question 5

What happens during the denaturation step of PCR?

Answer 5

The purified genomic DNA from the sample is placed in a micro centrifuge tube and heated for about 1 minute at 95 degrees celsius. This heating disrupts the hydrogen bonds holding the strands of the DNA double helix together, and the double-stranded DNA denatures into single-stranded DNA.

Question 6

What happens during the annealing step of PCR?

Answer 6

The two primers P1 and P2 anneal with the DNA.

Question 7

What are the two primers used in PCR?

Answer 7

They are called P1 and P2 (forward and reverse) that hybridize on either region we want amplified. The primers will amplify the DNA only in between the primers. Each primer set is designed to anneal to highly conserved regions of DNA within the barcode gene.

Question 8

What happens during the synthesis step of PCR?

Answer 8

Tae polymerase synthesizes DNA from the two primers.

Question 9

How many copies are generated during each cycle of PCR?

Answer 9

Each cycle generates two copies of the target fragment from a single template. Since the number of target fragments doubles with each cycle, the number of copies generated over multiple cycles is 2^n, where n equals the number of cycles. By carrying out multiple cycles in the thermal cycler, it is possible to obtain enormous levels of amplification.

Question 10

What is a conserved region of DNA?

Answer 10

A nucleotide sequence that has little to no variation across species- it has remained relatively constant throughout evolution.

Question 11

What is a master mix?

Answer 11

Instead of adding each reagant individually to each sample, you will make a PCR master mix.

Question 12

What does the master mix consist of?

Answer 12

The master mix consists of DNA polymerase, dNTPs, buffer, forward and reverse primers, and water in the quantity needed to perform multiple PCR reactions.

Question 13

What are the benefits of using a master mix?

Answer 13

A master mix helps reduce pipetting errors and time spent to set up the reaction.

Question 14

What is added to the sample after the master mix is added?

Answer 14

An aliquot.

Question 15

What is an aliquot?

Answer 15

A small portion of the master mix.

Question 16

What is agar?

Answer 16

a solid matrix of agarose and agaropectin that, with added nutrients, provides a growth medium for bacteria.

Question 17

What is direct amplification?

Answer 17

when the specific DNA fragments are amplified.

Question 18

What is the first step in barcoding?

Answer 18

digesting the organism

Question 19

What is the second step in barcoding?

Answer 19

purifying the DNA from the tissue, removing the proteins, RNA, and other cellular components before PCR. So, in other words, isolating the DNA.

Question 20

What is sterile technique?

Answer 20

involves a variety of procedure to prevent contamination of the solutions and cultures with which you are working.

Question 21

What is PCR?

Answer 21

a method used to amplify a specific fragment of DNA from a complex mixture of DNA.

Question 22

What is the PCR cycle?

Answer 22

95C (denature) –> 50C (anneal) -> 72C (synthesis)

Question 23

Why is Taq polymerase used?

Answer 23

Because most enzymes would degrade when at 95C. Taq polymerase is purified from Thermus aquaticus which is a bacterium that grows in hot springs. It does not degrade at 95C and 72C is the optimal temp for it to synthesize DNA.

Question 24

What happens during the denaturation step of PCR?

Answer 24

The purified genomic DNA from the sample is placed in a micro centrifuge tube and heated for about minute at 95C. This heating disrupts the hydrogen bonds holding the strands of the DNA double helix together, and the double-stranded DNA denatures into single-stranded DNA.

Question 25

What happens during the annealing step of PCR?

Answer 25

The two primers P1 and P2 anneal with the DNA.

Question 26

What happens during the synthesis step of PCR?

Answer 26

Allows Taq polymerase to synthesize DNA from the two primers.

Question 27

How many copies are generated during each cycle of PCR?

Answer 27

each cycle generates two copies of the target fragment from a single template. Since the number of target fragments doubles with each cycle, the number of copies generated over multiple cycles in 2^n, where n equals the number of cycles.

Question 28

What are 2 primers used in PCR?

Answer 28

They are called forward (For) and Reverse (Rev) that hybridize on either end of the region we want amplified. The primers will amplify only the DNA in between the primers. Each primer set is designed to anneal to highly conserved regions of DNA within the barcode gene.

Question 29

What is a conserved region of DNA?

Answer 29

a nucleotide sequence that has little to no variation across species - it has remained relatively constant throughout evolution.

Question 30

What marker is used for animal samples?

Answer 30

The COI gene that codes for cytochrome c oxidase is used as a marker for animal samples. It should generate around a 700 bp fragment.

Question 31

What marker is used for plant samples?

Answer 31

The rbcL gene found in the chloroplast genome and codes for ribulose-bisphosphate carboxylase is used as the marker for plant samples. It is often referred to as RuBisCo. The rbcL gene provides a DNA barcode marker to specifically identify plants and enables scientists to distinguish that plant from other organisms that may be living on the plant. It should generate around a 600 bp fragment.

Question 32

What marker is used for bacteria samples?

Answer 32

Since bacteria do not have mitochondria and chloroplasts another gene is used as a DNA barcode marker. The 16S rRNA gene that codes for the 16S ribosomal RNA that is part of the bacteria ribosome complex is used. It should generate around a 1500 bp fragment.

Question 33

What is a master mix?

Answer 33

Instead of adding each reagent individually to each sample, you can make a PCR master mix.

Question 34

What does the master mix consist of?

Answer 34

It consists of DNA polymerase, dNTPs, forward and reverse primers, and water in the quantity needed to perform multiple PCR reactions for a given primer set.

Question 35

What are the benefits of using a master mix?

Answer 35

A master mix helps to reduce pipetting errors and time spent running the reaction.

Question 36

What is added to the sample after the master mix is added?

Answer 36

An aliquot

Question 37

What is an aliquot?

Answer 37

a small portion of genomic DNA or bacterial to the PCR tube with the appropriate primer set.