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PCR and DNA Replication Essentials
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1
Question
What is the primary purpose of PCR?
Page 2
Answer
PCR is a technique to amplify a particular piece of DNA by making numerous copies of a segment.
2
Question
How many copies of a target DNA sequence can PCR produce in a few hours?
Page 2
Answer
Billions of copies.
3
Question
What does PCR stand for, and when was it invented?
Page 2
Answer
Polymerase Chain Reaction, invented in 1984.
4
Question
Why is PCR considered an integral part of molecular biology?
Page 2
Answer
Its applications are vast, allowing study of genes and DNA fragments.
5
Question
How does PCR relate to natural DNA replication?
Page 3
Answer
PCR is a laboratory version of DNA replication in cells.
6
Question
What do the terms 'in vitro' and 'in vivo' mean in the context of PCR and DNA replication?
Page 3
Answer
'In vitro' signifies occurring in a test tube; 'in vivo' signifies occurring in a living cell.
7
Question
What is the main difference in time efficiency between DNA replication in cells and PCR?
Page 4
Answer
DNA replication in cells takes hours to copy all of its DNA, while PCR can make billions of copies in a few hours.
8
Question
What is conservative replication in DNA?
Page 4
Answer
One strand of the original DNA serves as the template for the growth of a new DNA strand.
9
Question
How many errors occur on average during DNA replication in cells?
Page 4
Answer
Fewer than one error per billion nucleotides copied.
10
Question
How many enzymes and proteins participate in DNA replication in cells?
Page 4
Answer
More than a dozen.
11
Question
Name the key enzymes involved in DNA replication.
Page 4
Answer
DNA polymerase, DNA ligase, primase, helicase, topoisomerase, single-strand binding protein.
12
Question
In DNA replication, what is the leading strand?
Page 5
Answer
The leading strand is synthesized continuously in the 5' to 3' direction.
13
Question
What are Okazaki fragments in DNA replication?
Page 5
Answer
Okazaki fragments are short segments on the lagging strand that are later joined.
14
Question
What is the function of DNA polymerase in replication?
Page 6
Answer
It catalyzes the elongation of DNA by adding deoxyribonucleoside triphosphates to the 3' end of the growing strand.
15
Question
What is a deoxyribonucleoside triphosphate (dNTP)?
Page 6
Answer
It is a deoxyribose sugar + a base + three phosphate groups.
16
Question
How does energy from dNTPs contribute to DNA synthesis?
Page 6
Answer
When a dNTP joins the DNA strand, two phosphates are removed, and the energy is used to form the phosphodiester bond.
17
Question
What is the direction of DNA strand growth?
Page 6
Answer
DNA strands grow only by adding nucleotides to the 3' end.
18
Question
In DNA, what are the base-pairing rules?
Page 8
Answer
Adenine pairs only with thymine (2 H-bonds); guanine pairs only with cytosine (3 H-bonds).
19
Question
What is the structure of DNA?
Page 8
Answer
DNA is a double helix made up of nucleotides, with a sugar-phosphate backbone on the outside of the helix.
20
Question
How does DNA polymerase use each strand during replication?
Page 8
Answer
Once DNA strands are separated, each serves as a template to synthesize new strands of DNA with precise complementary sequences.
21
Question
What are the 5' and 3' ends in DNA strands?
Page 9
Answer
They refer to the numbers assigned to the carbons in the 5-carbon sugar; strands are oriented in opposite directions.
22
Question
What is the role of DNA ligase in replication?
Page 9
Answer
It joins Okazaki fragments on the lagging strand.
23
Question
Why can't DNA polymerase initiate DNA synthesis on its own?
Page 10
Answer
It can only add nucleotides to an existing 3' end of a primer.
24
Question
What is the function of primase?
Page 10
Answer
It creates a short RNA primer with a 3' end that DNA polymerase can add to.
25
Question
What happens to the RNA primer after replication?
Page 10
Answer
It is subsequently replaced with DNA.
26
Question
What does helicase do in DNA replication?
Page 11
Answer
It unwinds the two parallel DNA strands.
27
Question
What is the role of topoisomerase?
Page 11
Answer
It relieves the stress of twisting caused by helicase.
28
Question
What do single-strand binding proteins do?
Page 11
Answer
They bind to and stabilize the unpaired DNA strands.
29
Question
What are the main components needed to perform PCR?
Page 12
Answer
1. DNA of interest with target sequence; 2. DNA polymerase (like Taq); 3. dNTPs; 4. Buffers; 5. Primers; 6. Thin-walled tubes; 7. Thermal cycler.
30
Question
Why are thin-walled tubes used in PCR?
Page 13
Answer
They allow for low volume, low cost, and reduced evaporation and heat transfer concerns.