Topic 11 - Replication & PCR

Question 1

What type of replication does DNA undergo?

Answer 1

Semi-Conservative

Question 2

What does semi-conservative replication mean?

Answer 2

a permanent separation
between the two strands of DNA, and each isused as a template to produce “new” DNA strand

Question 3

What are the two new double-stranded DNA molecules produced called?

Answer 3

Daughter Molecules

Question 4

What are the Four-Step Process of Replication

Answer 4

Step 1: Separation of the Strands

Step 2: Short RNA primer is formed

Step 3: Use both strands of DNA as a template

Step 4: RNA is replaced and replication ends

Question 5

Enzymes to look out for in step 1

Answer 5

Helicase, Topoiosomerase

Question 6

Enzymes to look out for in step 2

Answer 6

Primase

Question 7

Enzymes to look out for in step 3

Answer 7

DNA polyamerase

Question 8

Enzymes to look out for in step 4

Answer 8

Nucease, DNA

Question 9

Where does separation of the two DNA strands occur?

Answer 9

Separation occurs at various points along the chromosome called origins of replication.

Question 10

What is created when strands separate at the origins of replication?

Answer 10

multiple replication “bubbles” are created

Question 11

What forms as the separation stretches away from the origin?

Answer 11

A replication fork

Question 12

Which protein keeps separating the two DNA strands?

Answer 12

Helicase protein

Question 13

What is the role of Single-Strand Binding Proteins (SSBP)?

Answer 13

They bind to the single strands of DNA to keep them from reannealing

Question 14

What is the function of Topoisomerase during replication?

Answer 14

Removes DNA twisting tension by temporarily breaking a phosphodiester bond, rotating the strand to relieve tension, and then rejoining the DNA.

Question 15

Why can’t DNA polymerase start building a new DNA strand from single-stranded DNA (ssDNA)?

Answer 15

Because DNA polymerase can only add new monomers to the 3’ end of an existing strand.

Question 16

Which enzyme attaches to ssDNA and synthesizes a short RNA primer?

Answer 16

Primase.

Question 17

In what direction does Primase synthesize RNA primers?

Answer 17

5’ -> 3’ direction, moving in a 3’ -> 5’ direction on the template

Question 18

Which enzyme attaches to the RNA primer and produces the complementary strand?

Answer 18

DNA Polymerase III

Question 19

In what direction does DNA Polymerase III move on the template strand?

Answer 19

moving 3’ -> 5’ on the template, and building the new daughter strand in a 5’ ->3’ direction

Question 20

How many DNA strands are used as templates during replication?

Answer 20

BOTH DNA strands are “templates”

Question 21

What is the difference between the leading and lagging strands?

Answer 21

The leading strand:
is synthesized continuously

lagging strand: is synthesized discontinuously.

Question 22

Which enzyme replaces the RNA primers with DNA?

Answer 22

DNA polymerase 1 (which can also act as a nuclease)

Question 23

What is the final enzyme involved in replication, and what does it do?

Answer 23

ligase forms phosphodiester bonds between the newly
synthesized section of DNA
and the rest of the DNA
strand.

Question 24

How often do initial pairing errors occur between incoming nucleotides and the template strand?

Answer 24

At a rate of 1 in 10⁵ nucleotides.

Question 25

How often do errors remain in the completed DNA molecule?

Answer 25

About 1 in 10¹⁰ nucleotides.

Question 26

What makes this reduction in error rate possible?

Answer 26

During replication, DNA polymerase and other proteins verify the integrity of the nucleotide sequence being copied.

Question 27

Why are mutations important?

Answer 27

Because mutations create alleles, which are important for evolution.

Question 28

What is the Polymerase Chain Reaction (PCR)?

Answer 28

allow for the rapid and specific replication of a portion of DNA, without the need for a cell

Question 29

How does PCR help scientists study specific regions of DNA?

Answer 29

PCR allows targeting of a specific region on a chromosome and amplifies it (makes many copies) so that machines can read it.

Question 30

What does DNA polymerase need to attach to the DNA template and begin adding nucleotides?

Answer 30

DNA polymerase needs a primer to attach and begin adding nucleotides to the 3’ end.

Question 31

How are DNA primers positioned in PCR?

Answer 31

DNA primers are placed on each side of the target DNA region, surrounding it so that the section in between can be copied.

Question 32

What type of primers are used in PCR?

Answer 32

DNA primers are used in PCR — not RNA primers.

Question 33

What happens during Step 1 of PCR?

Answer 33

The double-stranded DNA (dsDNA) is denatured (separated).

Question 34

How is the DNA denatured in PCR?

Answer 34

By heating to about 95°C, which increases kinetic energy and breaks the hydrogen bonds between complementary bases.

Question 35

What happens to the DNA strands after heating?

Answer 35

The dsDNA separates into single strands.

Question 36

Are enzymes like helicase or topoisomerase needed in PCR?

Answer 36

No, because heat separates the strands, so these enzymes are not required.

Question 37

What happens during Step 2 of PCR?

Answer 37

The system is cooled(50-60) so that the short DNA primers can bind to the desired region(targeting)

Question 38

Why doesn’t the full DNA molecule reanneal during primer binding?

Answer 38

1. Because the reaction mixture contains primers at a much higher molar concentration than the DNA template. 2. The annealing step is short (20–40 seconds), and full-length DNA strands take longer to reanneal due to their length and complexity.

Question 39

What enzyme copies the DNA template strands during elongation?

Answer 39

Taq polymerase, a special DNA polymerase

Question 40

Where does Taq polymerase come from?

Answer 40

It comes from a bacterium called Thermus aquaticus, which is adapted to live in very hot environments.

Question 41

At what temperature does Taq polymerase work best?

Answer 41

72°C, which allows it to replicate DNA without the two strands reannealing.

Question 42

What does Taq polymerase need in order to synthesize new DNA strands?

Answer 42

1.Deoxyribonucleotides (dNTPs) 2. Mg²⁺ ions

Question 43

What is the role of Mg²⁺ ions in PCR?

Answer 43

Mg²⁺ acts as a cofactor, helping the enzyme catalyze the addition of dNTPs to the growing DNA strand.

Question 44

How many DNA molecules are made after the 1st round of PCR, and what’s their length?

Answer 44

2 molecules are made, and they are too long (longer than the target sequence).

Question 45

How many DNA molecules are made after the 2nd round of PCR, and what’s their length?

Answer 45

2nd round makes 4 molecules (still too long)

Question 46

How many DNA molecules are made after the 3rd round of PCR, and how many are the desired sequence?

Answer 46

3rd round makes 8 molecules (2 are just desired sequence)

Question 47

How many DNA molecules are made after the 4th round of PCR, and how many are the desired sequence?

Answer 47

4th round makes 16 molecules (1/2 are now desired sequence)

Question 48

After 25–30 rounds of PCR, what percentage of the DNA is the desired sequence?

Answer 48

About 99.99% is the desired sequence.