Genetics Flashcards
DNA structure
double helix - double stranded anti parallel configuration 3’ 5’ A-T 2 hydrogen bonds C-G 3 hydrogen bonds hydrophillic phosphate deoxyribose backbone
Helicase / Topoisomerase
unzip and unwind DNA at replication forks
origins of replications
often in AT regions (only 2 hydrogen bonds) for Eukaryotes there are multiple as they have lots of DNA but prokaryotes only have a single OR.
Primase
is an enzyme that synthesizes short RNA sequences called primers. These primers serve as a starting point for DNA synthesis. error prone
DNA polymerase III
DNA polymerase 3 is essential for the replication of the leading and the lagging strands.fast 5’-3’ polymerase, 3’-5’ exonuclease activity. can proof read too
DNA polymerase I
DNA polymerase 1 is essential for removing of the RNA primers from the fragments and replacing it with the required nucleotides. slow 5’-3’ polymerase, 3’-5’ and 5’-3’ exonuclease activity
DNA Ligase
joins two DNA fragments by forming new phosphodiester bonds. It is used in cells to join together the Okazaki fragments which are formed on the lagging strand during DNA replication.
DNA replication steps
Step 1: DNA unzipping (DNA topoisomerase, helicase)
Step 2: RNA primer synthesis (DNA primase)
Step 3: base pairing (DNA polymerase)
Step 4: phosphodiester bonding between nucleotides
(DNA ligase)
Transcription
Synthesis of mRNA by RNA polymerase
EUKARYOTES vs PROKARYOTES
DNA in the nucleus transcription occurs inside nucleus then exported in cytoplasm for translation
everything occurs in cytoplasm
Constitutive promoter
e.g. tRNA,s, rRNAs, ribosomal proteins,
RNA polymerase
Inducible promoters
Promotors subject to up/down regulation (Lac, Pho
operons)
Transcription (σ) factors
- protein that binds to promoter region, thereby initiating transcription
Promoter
specific non-coding DNA sequences to which RNA polymerase holoenzyme binds
Enhancer / silencer
regions of DNA (distal) that bind trans- acting factors to enhance/repress transcription
Operator
segment of DNA to which a repressor binds (thereby blocking RNApol binding)
Regulon
a group of genes regulated by the same regulatory molecule; may be located non-contiguously on the genome
Translation
Step 1: an amino-acyl tRNA binds to a vacant A-site, a spent tRNA dissociates from the E-site
Step 2: a new peptide bond is formed
Step 3: the large ribosome subunit translocates, leaving the 2 tRNAs in hybrid sites
Step 4: the small ribosome subunit translocates,
carrying its mRNAs a distance of 3 nucleotides through the ribosome. The ribosome is “reset” for a next cycle.
TRANSFER RNA (tRNA)
tRNAs are covalently bound to amino-acids
via aminoacyl-tRNA synthetase
•tRNA match amino-acids to codons in mRNA
Recombinant DNA cloning
Step 1: isolate genomic DNA from organism
Step 2: Amplify target DNA via PCR (and purify) Step 3: Cleave target and plasmid DNA with restriction endonucleases to create ‘sticky ends’ Step 4: Ligate DNA fragments into a cloning vector = create a recombinant plasmid
Step 5: Transform recombinant plasmid into host system that will replicate
PCR- Polymerase Chain Reaction
Molecular method to exponentially amplify DNA using a thermostable DNA polymerase and thermal cycles
PCR cycle
- DENATURATION
(melting) 1 min at 95oC - PRIMER ANNEALING
2 to 60s at 56-64 °C - EXTENSION
4 to 120s at 72 °C
Quantitative PCR
In qPCR an fluorescent dye (SYBR) or probe (Taqman) is used to determine the number of amplicons after each PCR cycle. Principle: use of a fluorescent “reporter” bound to a “quencher”
Plasmids
circular episomal (extra-chromosomal) dsDNA molecules
Transformation
The uptake and incorporation of exogenous genetic material through the cell membrane. For transformation to occur, recipient cells must be in a state of competence.
Transduction
is the process by which DNA is transferred from one bacterium to another via bacteriophage (virus). Different methods to perform DNA uptake; the most common:
chemoporation (heat shock), electroporation (electric pulses), conjugation, transduction.
Bacterial Conjugation
Exchange of genetic material between bacteria. Requires physical contact (DNA exchanged via Pilli)
screening vs selection
selection - defined the ones which have the plasmid (determined from colour blue = insert not present, white = insert present)
Screening - find the ones with the genes of interest
Plasmid instability occurs as a result of:
- DNA mutation (inactivation)
- Defective plasmid segregation (via low copy number)
- Insufficient selective pressure (e.g. no antibiotics)
Restricition endonucleases
Restriction enzyme, also called restriction endonuclease, is a protein produced by bacteria that cleaves DNA at specific sites along the molecule. Cleave target and plasmid DNA with restriction endonucleases to create ‘sticky ends’
Plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria
RNA polymerase
RNA polymerase uses one of the DNA strands (the template strand) as a template to make a new, complementary RNA molecule
