L1-3: Molecular Cloning Flashcards
Green Fluorescent Protein (GFP)
Unique protein produced by jellyfish Aequorea victoria that emits green colour in blue/UV light
Basic components of DNA
Phosphate backbone, deoxyribose sugar, nucleotide bases
Structure of DNA
Antiparallel helix with deoxyribose sugars linked together through phosphodiester bonds.
T4 DNA Ligase
- DNA ligase frombacteriophage T4
- Catalyses the formation of a phosphodiester bond between 5’ phosphate and 3’ hydroxyl termini in duplex DNA or RNA
- It can ligate eithercohesive (sticky) or blunt endsof DNA, oligonucleotides, as well as RNA and RNA-DNA hybrids, but not single-stranded nucleic acids
- Ligatesblunt-ended DNAwith much greater efficiency thanE. coliDNA ligase
DNA Gyrase
- Enzyme within the class of topoisomerase, subclass of type II topoisomerase
- Reduces topological strain in ATP-dependent manner while dsDNA is being unwound by elongating RNA polymerase or by helicase in front of the progressing replication fork
Is the origin of replication A/T or G/C rich?
A/T
DNA primase synthesises the RNA primer in a ____ direction.
5’ to 3’
Continuous DNA synthesis occurs in the ____ strand.
leading
Main differences between prokaryotes and eukaryotes
Prokaryotes: lack nucleus and organelles; single-celled; single circular chromosome
Eukaryotes: contain nucleus, and organelles; can be single-celled or multicellular; multiple linear chromosomes; much larger than prokaryotes
Cell compartments containing DNA in eukaryotes
- Nucleus
- Mitochondria
- Chloroplasts (plant cells)
T or F: only eukaryotes contain ribosomes
False.
T or F: only eukaryotes contain a cytoplasm
False.
T or F: prokaryotes do not have a plasma membrane
False.
Restriction endonucleases
Recognise and cut dsDNA at specific sequences called restriction/recognition sites
Endonucleases
Cleave within the DNA molecule at internal phosphodiester bonds
Exonucleases
Cleave at ends of DNA molecule
Recognition/restriction site
Palindromic nucleotide sequence that is recognised
Cleavage site
Phosphodiester bond that is cleaved
Frequency of restriction sites
4^n, where n = number of recognisable base pairs in the restriction site sequence
Restriction modification system
The system bacteria possess for defence against its own REs. This system is composed of a restriction endonuclease enzyme and a methylase enzyme. Each bacterial species and strain has their own combination of restriction and methylating enzymes.
Methylase/methyl transferase - an enzyme that adds a methyl group to a molecule; in restriction-modification systems of bacteria a methyl group is added to DNA at a specific site to protect the site from restriction endonuclease cleavage.
Isoschizomer
REs specific to the same recognition site e.g. Sph I, Pae I, Bbu I (5’-CGTAC/G-3’).
Isoschizomers are isolated from different strains of bacteria and therefore may require different reaction conditions.
Neoschizomer
Recognise the same sequence but cut it differently e.g. Sma I (5’-CCC/GGG-3’) and Xma I (5’-C/CCGGG-3’)
Isocaudomer
recognise slightly difference sequence, but produces same ends e.g. Mbo I (5’-N/GATCN-3’) and BamIH I (5’-G/GATCC-3’)
Klenow fragment
DNA Polymerase I, Large (Klenow) Fragment lacks the 5’ to 3´ exonuclease activity of intact E. coli DNA polymerase I, but retains its 5´ to 3´ polymerase, 3´ to 5´ exonuclease and strand displacement activities.
DNA is ____ charged
negatively