Chapter 12

Question 1

What is DNA made of?

Answer 1

• Sugar-phosphate backbones on outside of double helix, nitrogenous bases
• DNA strands antiparallel from 5’ -> 3’

Question 2

Denature vs Renature

Answer 2

Denature: heat or chemicals can denature or break apart DNA to single strands

Renature: cooling or removal of chemical denaturants can allow DNA strands to reanneal.

Question 3

RNA polymerase

Question 4

DNA Ligase

Question 5

DNA Polymerase I

Question 6

Primase

Question 7

DNA Polymerase III

Answer 7

DNA-Dependent DNA polymerase that copies ssDNA starting from a “dsDNA” region. Direction of synthesis is 5’ -> 3’

• has been inactive/denatured by the high temperature, thus one have to add DNA pol II again.

Question 8

Helicase

Question 9

Topoisomerase

Question 10

What is PCR

Answer 10

Polymerase Chain Reactions

Question 11

Primers/Oligonucleotides

Answer 11

-
- To design primers, one need to know the DNA sequence!

Question 12

When is Taq DNA pol most active or stable

Answer 12

Taq DNA pol most active @ ~72C & remains stable at 95C

Question 13

What is the process of PCR?

Answer 13

Reaction Mix: Template DNA, dNTPs, Taq DNA pol, 2 primers x-template DNA mol
- Denaturation (95C ~1min)

    - Annealing/Hybridization (variable T ~1min)

    - Elongation/Polymerization (72C, 1min/kbp) 2x-DNA mol

    - Denaturation (95C ~1min)

    - Annealing/Hybridization (variable T ~1min)

    - Elongation/Polymerization (72C, 1min/kbp) 4x- DNA mol

    - Denaturation (95C ~1min)

Question 14

What is PCR use for?

Answer 14

Detecting specific sequences and copying DNA for recombinant DNA tech/biotech
- Detection of RNA (RT-PCR- Covid19)

Question 15

What is Reverse Transcriptase?

Answer 15

Viral enzyme that synthesize some ssDNA starting from ssRNA used during retroviral reverse transcription

• synthesize the cDNA from 5’ -> 3’ while moving on the template RNA from 3’ -> 5’

Reaction Mix: Template DNA, dNTPs, Taq DNA pol, 2 primers x-template DNA mol
- Denaturation
- Annealing/Hybridization
- Elongation/Polymerization (complimentary ds-
DNA c-dsDNA)
- Denaturation (95C ~1min)
- Annealing/Hybridization (variable T ~1min)

Question 16

RT-PCR for Eukaryotic mRNA

Answer 16

mature RNA have:
- 3’ poly tail
- 5’ cap
- spliced (introns removed)
- encodes proteins
- found in cytoplasm for easy extract

Question 17

What is important for mRNA

Answer 17

• 3’ poly-A tail can be targeted with oligo-dT (a primer made of Ts) and form there, one can amplify the coding sequence of Eukaryotic genes as cDNA (with no introns)

Question 18

PCR v.s. RT-PCR

Answer 18

• regular PCR for DNA genes = has introns (not coding for proteins
• RT-PCR for mRNA genes = coding sequence only (no introns)

Question 19

What are restriction enzymes?

Answer 19

• endonuclease that recognize specific garget nucleotide sequence in DNA and breaks the DNA at those points