DNA Replication

Question 1

What does PCR stand for and what does it mean?

Answer 1

Polymerase Chain Reaction, = synthesis of
DNA by thermostable
DNA polymerase in the
test tube

Question 2

Define DNA Cloning

Answer 2

Prior to the 1980’s, the primary method for producing many copies of a gene

Question 3

What is the key element of PCR?

Answer 3

Heat. DNA is submitting through heating and cooling cycles.

Question 4

Explain the steps of PCR and how it works

Answer 4

Denaturation, Annealing, and Primer Extension. GATHER, BOUND, POLYMERASE, TO MIMIC THE PROCESS OF DNA REPLICATION First, a small amount of DNA is gathered, a pair of primers are used to bind each end of the target sequence, Then a dna polymerase is used, then Four dNTPs (i.e., dATP, dCTP, dGTP, dTTP), and last a few essential ions and salts. They use those ingredients to to mimic the natural dna replication process. A thermocycler is used with cool and heat.

Question 5

What is a Thermocycler?

Answer 5

A machine that jump-starts each stage of the reaction by raising and lowering the temperature of the chemical components at specific times and for a preset number of cycles.

Question 6

What Denatursation?

Answer 6

The melting of DNA into individual strands. The started solution is heated to break the bonds joining the strands of a DNA double helix enabling it to separate into two strands.

Question 7

What is Annealing?

Answer 7

The reaction mixture is quickly cooled, usually between 30 and 65 degrees. This gives the primers an opportunity to bind or anneal to their complementary sequences on the single strands of DNA

Question 8

What is Extension?

Answer 8

The sample is heated and the DNA polymerase begins to make a new DNA strand by attaching to the primers

Question 9

What is (RT-PCR)?

Answer 9

Reverse Transcription Polymerase Chain Reaction, this combines real time PCR and reverse transcription

Question 10

What is Reverse Transcription?

Answer 10

The process that makes dna from rna

Question 11

What is qPCR?

Answer 11

quantitative real-time PCR (qPCR)

Question 12

What is PCR used for?

Answer 12

Sequencing, cloning, and analysis in the case of forensics

Question 13

What are the two primers called?

Answer 13

Forward and Reverse primers

Question 14

What can we tell if both primers attach to the same piece of DNA and synthesize in the same direction?

Answer 14

That there is no PCR product

Question 15

Explain the process of replication

Answer 15

Template strands separate, New nucleotides are added according to the templates, and they are joined into the new strand

Question 16

T/F Replication occurs prior to cell division

Answer 16

True

Question 17

T/F Replication happens during the G1 phase of the cell cycle

Answer 17

False. It happens during the S phase.

Question 18

T/F The cell divides producing
two daughter cells during the
mitotic phase

Answer 18

True

Question 19

In the cell, splitting is performed by a protein called what

Answer 19

Helicase

Question 20

What temperature does the helicase unwind the DNA helix at?

Answer 20

37 degrees which is body temperature

Question 21

What are origins of replication?

Answer 21

The origin is a specific sequence in the DNA that is recognized
by DNA polymerase as a start point for replication

Question 22

The origin is usually an AT rich region, because

Answer 22

AT regions are easier to break.

Question 23

T/F There is only one
origin of replication
in a single
chromosome

Answer 23

False. There are many origins in a single chromosome.

Question 24

T/F Each strand can provide a new template for building a new strand

Answer 24

True

Question 25

T/F New nucleotides are arranged in the new strand by their ability to complement the template strand bases

Answer 25

True!

Question 26

T/F G’s bind with A’s * A’s bind with C’s

Answer 26

False - G’s bind with C’s * A’s bind with T’s

Question 27

T/F DNA polymerase adds about 1000-5000 bp/minute depending on conditions

Answer 27

True

Question 28

T/F Replication can occur in many directions

Answer 28

False. It can only occur in one direction

Question 29

DNA polymerase can start a new strand by itself

Answer 29

False. it cannot start a new strand by itself. It only can add to an existing strand

Question 30

What is primase?

Answer 30

An enzyme consisting of RNA is added to a sequence

Question 31

What is a primer?

Answer 31

In the cell, a short primer sequence consisting of RNA is added by a enzyme called Primase.

Question 32

T/F Since the primer is actually made of RNA nucleotides, it must be eventually removed and replaced with DNA.

Answer 32

True!

Question 33

T/F The leading strand has a primer at the beginning, and replication occurs continuously as the DNA unwinds.

Answer 33

True!

Question 34

T/F The lagging strand grows in the same direction from the movement of helicase through the production of short segments of DNA (Okazaki fragments).

Answer 34

False. It is the opposite direction.

Question 35

T/F The enzyme ligase seals the Okazaki fragments together after removal of the primers.

Answer 35

True

Question 36

T/F Before editing the error rate is about 1/100,000.

Answer 36

True

Question 37

After editing the error rate in replication is about 1/1,000,000

Answer 37

False! It's After editing the error rate in replication is about 1/10,000,000

Question 38

T/F These errors create mutations in the DNA from one generation to the next

Answer 38

True!

Question 39

Are Mutations in the DNA the source of variation in living organisms?

Answer 39

YES!

Question 40

What are the four components required for PCR?

Answer 40

Template DNA (to be copied). (primer, polymerase, dntp, thermocycler) 2. Primers (to initiate the reaction) 3. DNA polymerase (to add the bases) 4. dNTP (deoxyribonucleoside triphosphates) or the A’s, T’s, C’s, and G’s 5. While PCR can be done with heat plates and ice baths, all labs now simply use Thermal Cyclers.

Question 41

What is the temperature for Denature?

Answer 41

94 degrees

Question 42

What is the temperature for Anneal?

Answer 42

30-65 degres, it's more variable

Question 43

What is the temperature for extension ?

Answer 43

72 degrees

Question 44

Why do our cells require helicase, but we can get by without it during PCR?

Answer 44

Our cells require helicase instead of denaturation because if we were to heat our bodies up to 94-95 C, we would die. Plain and simple. That sort of extreme heat would cause all of our systems to shut down, and cells to commit to apoptosis (cell death). With PCR we are allowed a certain leniency with environmental controls. Because of this, we can go through cycles of denaturing and annealing the DNA in order to better control our product yield. Furthermore, with PCR, you can increase and decrease the temperature to allow for multiple reactions to occur. However, with helicase, once you add the enzyme to the PCR reaction, it will unzip all strands and keep doing so – even the strands we want to keep together.

Question 45

T/F Amplification always occurs in the 5’ 3’ direction!!

Answer 45

True

Question 46

If you start with 1 double-stranded DNA molecule as a template and conduct a PCR for 5 thermal cycles, how many DNA molecules of the PCR product will you synthesize at the end of the reaction?

Answer 46

32. If starting with 1 molecule, the final number of double stranded DNA molecules can be calculated using 2n 1 double-stranded DNA = 2n = 25 = 32

Question 47

What side does a primer need to bond onto DNA?

Answer 47

The 3 side, never the 5