What is DNA made of?
- Sugar-phosphate backbones on outside of double helix, nitrogenous bases
- DNA strands antiparallel from 5’ -> 3’
Denature vs Renature
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.
RNA polymerase
DNA Ligase
DNA Polymerase I
Primase
DNA Polymerase III
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.
Helicase
Topoisomerase
What is PCR
Polymerase Chain Reactions
Primers/Oligonucleotides
-
- To design primers, one need to know the DNA sequence!
When is Taq DNA pol most active or stable
Taq DNA pol most active @ ~72C & remains stable at 95C
What is the process of PCR?
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)What is PCR use for?
Detecting specific sequences and copying DNA for recombinant DNA tech/biotech
- Detection of RNA (RT-PCR- Covid19)
What is Reverse Transcriptase?
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)
RT-PCR for Eukaryotic mRNA
mature RNA have:
- 3’ poly tail
- 5’ cap
- spliced (introns removed)
- encodes proteins
- found in cytoplasm for easy extract
What is important for mRNA
- 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)
PCR v.s. RT-PCR
- regular PCR for DNA genes = has introns (not coding for proteins
- RT-PCR for mRNA genes = coding sequence only (no introns)
What are restriction enzymes?
- endonuclease that recognize specific garget nucleotide sequence in DNA and breaks the DNA at those points
