1. Mutation
1.1. Location
1.1.1. Coding sequence
1.1.2. Promoter
1.1.3. Intron
1.1.4. Intergenic
1.2. Type
1.2.1. First mutation
1.2.1.1. Large scale
1.2.1.1.1. duplication
1.2.1.1.2. inversion
1.2.1.1.3. rearragement
1.2.1.2. Single base pair
1.2.1.2.1. Silent
1.2.1.2.2. Nonsense (stop codon)
1.2.1.2.3. Missense
1.2.1.3. Indels
1.2.1.3.1. Altered reading frame (frameshift)
1.2.2. Second mutation
1.2.2.1. Supression mutation
1.2.2.1.1. Reversion
1.2.2.1.2. Intragenic
1.2.2.1.3. Extragenic
1.3. Consequence
1.3.1. Lose of gene function
1.3.2. Gain of gene function
1.3.3. No effect
1.3.4. Dominant negative function
1.4. classification based on cause
1.4.1. Spontatenous mutation
1.4.1.1. DNA replication error
1.4.1.1.1. Base pair substitution
1.4.1.1.2. Slippage during DNA replication
1.4.1.2. Damage to DNA
1.4.1.2.1. depurination
1.4.1.2.2. deamination
1.4.1.2.3. oxidation
1.4.1.2.4. strand breakage
1.4.2. Induced mutation
1.4.2.1. by mutagens
1.4.2.1.1. Replace normal base pair
1.4.2.1.2. Specific mispairing
1.4.2.1.3. loss of pairing
1.4.2.1.4. chromosome breaks
2. Repair
2.1. Mechanism
2.1.1. Reversal DNA damage
2.1.1.1. phr gene (photoreactivation)
2.1.1.2. ada gene (methyl transferase)
2.1.2. Removal DNA damage
2.1.2.1. nucleotide excision repair
2.1.2.1.1. uvr excision
2.1.2.1.2. DNA glycosylase and AP endonuclease
2.1.3. Post replication repair
2.1.3.1. mismatch repair
2.1.3.2. Sos repair
2.1.4. Double stranded break repair
2.1.4.1. Non homologous end joining (no template)
2.1.4.2. Homologous end joining (need template)
3. Gene cloning
3.1. How to do gene cloning
3.1.1. In vitro
3.1.1.1. PCR
3.1.2. Invivo
3.1.2.1. recombinant DNA
3.1.2.1.1. How?
3.2. Types of DNA library
3.2.1. genomic
3.2.1.1. how to make?
3.2.1.1.1. take genomic DNA, cut with Restriction enzyme (partial or complete) then clone it into vector
3.2.2. cDNA
3.2.2.1. how to make?
3.2.2.1.1. take RNA, copy mRNA to DNA, ligate into suitable vector
3.3. Types of Library vector
3.3.1. plasmid
3.3.1.1. size of insert: up to 8 kb
3.3.2. bacterophage
3.3.2.1. size insert up to 40 kb
3.3.2.2. need packaging
3.3.3. Fosmid
3.3.3.1. size insert up to 35 - 45 kb
3.3.3.2. hybrid between phage and F factor plasmid
3.3.3.3. need packaging
3.3.4. Bacterial arteficial chromosome
3.3.4.1. size insert up to 100 to 150 kb
3.3.4.2. no need packaging
3.3.5. yeast artificial chromosome
3.3.5.1. size insert up to 500 kb
3.3.5.2. make in yeast
3.4. Types of library screening
3.4.1. By function (phenotype)
3.4.2. By sequence homology (probe)
3.4.3. By protein recognition (antibody)
3.4.4. By sequencing
3.4.4.1. Sanger sequencing
3.4.5. Mapped based cloning approach
3.4.5.1. chromosomal walking
3.4.5.2. chromosomal jumping
3.5. Types of blotting
3.5.1. Southern blotting - DNA
3.5.2. Northern blotting - RNA
3.5.2.1. high strigency for mRNA abundance
3.5.2.2. low strigency for mRNA of different species
3.5.3. Westren blotting - protein
3.6. Genetic maps
3.6.1. Molecular marker
3.6.1.1. Single nucleotide polymorphism
3.6.1.1.1. detect by sequencing
3.6.1.1.2. detect as restriction fragment length polymorphism using DNA hybridization
3.6.1.2. Simple sequence length polymorphism
3.6.1.2.1. usually to detect microsatelite
3.6.1.2.2. detect by PCR
3.6.1.2.3. detect by DNA hybridization
4. Gene maintenance and expression
4.1. DNA replication
4.1.1. Prokaryotic
4.1.1.1. DNA replication is semi conservative
4.1.1.2. DNA polymerase synthesize from 5' to 3'
4.1.1.3. DNA replication requires double stranded template
4.1.1.4. Components
4.1.1.4.1. Initiator protein
4.1.1.4.2. Helicases
4.1.1.4.3. SSB proteins
4.1.1.4.4. DNA gyrase
4.1.1.4.5. Primase
4.1.1.4.6. DNA polymerase III
4.1.1.4.7. DNA polymerase I
4.1.1.4.8. DNA ligase
4.1.2. Eukaryotic
4.1.2.1. DNA replication is semi conservative
4.1.2.2. DNA polymerase synthesize 5' to 3'
4.1.2.3. requires double stranded template
4.1.2.4. Components
4.1.2.4.1. Initiator protein
4.1.2.4.2. Helicases
4.1.2.4.3. SSB proteins
4.1.2.4.4. DNA gyrase
4.1.2.4.5. Primase complex (DNA polymerase, DNA pol alpha-primase)
4.1.2.4.6. DNA polymerase delta
4.1.2.4.7. DNA polymerase epsilon
4.1.2.4.8. DNA polymerase gamma
4.1.2.4.9. DNA ligase
4.2. Transcription
4.2.1. Prokaryotic
4.2.1.1. Initiation: at -10 recognition (Pribnow box) and -35 recognition
4.2.1.2. Elogation: All genes by single RNA polymerase with sigma factor
4.2.1.3. Termination:
4.2.1.3.1. Rho independent (GC rich)
4.2.1.3.2. Rho dependent
4.2.2. Eukaryotic
4.2.2.1. Initiation: 5 Cap
4.2.2.2. Elogation: Multiple RNA polymerases
4.2.2.3. Termination: Poly A tail
4.2.2.4. Post-transcriptional modification
4.2.2.4.1. Splicing:
4.2.3. Type of rRNA in eukaryotic
4.2.3.1. rRNA
4.2.3.1.1. required for translation
4.2.3.1.2. final product of gene expression
4.2.3.1.3. not translated
4.2.3.1.4. stable
4.2.3.2. protein coding (mRNA)
4.2.3.2.1. translated into polypeptide
4.2.3.2.2. intermediate of gene expression
4.2.3.2.3. unstable
4.2.3.3. tRNA
4.2.3.3.1. required for translation
4.2.3.3.2. wooble / nondegeneracy
4.2.3.3.3. final product of gene expression
4.2.3.3.4. not translated
4.2.3.3.5. stable
4.3. Translation
4.3.1. Prokaryotic cells
4.3.1.1. Initiation
4.3.1.1.1. Starts with Shine Dalgarno sequence
4.3.1.1.2. initiator tRNA fMET (AUG start site)
4.3.1.2. Elogation
4.3.1.2.1. E-P-A site
4.3.1.3. Termination
4.3.1.3.1. release factor (RF 1 and RF 2) recognizing stop codon
4.3.2. Eukaryotic cells
4.3.2.1. Initiation
4.3.2.1.1. Cap binding protein (eIF4F)
4.3.2.1.2. initiator tRNA MET (AUG start site)
4.3.2.2. Elogation
4.3.2.2.1. E-P-A site
4.3.2.3. Termination
4.3.2.3.1. stop codon
5. Characteristic of DNA, Gene and Genome
5.1. Characteristic of DNA
5.1.1. double helix
5.1.2. anti parallel
5.1.3. phosphate sugar backbond
5.1.4. nucleotide inside
5.1.5. carries genetic code
5.2. Characteristic of genome
5.2.1. prokaryotic
5.2.1.1. chromosomal DNA
5.2.1.1.1. usually circular
5.2.1.1.2. located in nucleoid (membraneless)
5.2.1.1.3. supercoil
5.2.1.1.4. low repetitive DNA
5.2.1.1.5. high density protein coding gene
5.2.1.2. extra chromosomal DNA (plasmid)
5.2.1.2.1. low to high copies number (variable)
5.2.1.2.2. generally dispensable for host function unlike chromosomal DNA
5.2.1.2.3. transfer from cells to cells (cross species)
5.2.2. eukaryotic
5.2.2.1. Nuclear DNA
5.2.2.1.1. Located in nucleus
5.2.2.1.2. Linear
5.2.2.1.3. Present as one, two or multiple copy (haploid, diploid etc)
5.2.2.1.4. Chromosome number does not relate to complexity
5.2.2.1.5. Associated with protein (histone)
5.2.2.1.6. Structure
5.2.2.2. Mitochondrial DNA
5.2.2.2.1. Located in mitochodnria
5.2.2.2.2. Mostly circular
5.2.2.2.3. DNA double stranded and supercoil
5.2.2.2.4. Genetic variation in size
5.2.2.3. Chroloplast DNA
5.2.2.3.1. Located in chloroplast
5.2.2.3.2. All genome circular
5.2.2.3.3. DNA double strand and supercoil
5.2.2.3.4. Generally similar in size
5.2.3. HUMAN genome
5.2.3.1. Intergenic DNA (highest in composition)
5.2.3.1.1. Interspersed repeats
5.2.3.1.2. Other intergenic region
5.2.3.2. Gene and related sequence
5.2.3.2.1. Exon (lowest in composition)
5.2.3.2.2. Related sequence
5.3. Variety in genome
5.3.1. Base composition (GC content)
5.3.1.1. DNA melt curve
5.3.2. Complexity
5.3.2.1. reassociation kinetics
5.3.3. Composition
5.3.3.1. DNA hybridization and gene cloning
5.3.4. Size
5.3.4.1. Quantification of phosphate, re association kinetic, flow cytometry
6. Genomic study and evolution
6.1. Identifying ORF
6.1.1. contain ATG
6.1.2. contain termination codon
6.1.3. codon bias
6.1.4. exon intron boundary
6.1.5. Tata box
6.2. Functional and comparative genomic
6.2.1. Homology
6.2.1.1. Orthology
6.2.1.2. Paralogy
6.2.2. How to study function of gene
6.2.2.1. GEnome wide screen
6.2.2.1.1. insertional inactivation
6.2.2.1.2. knockdown using RNA interference
6.2.2.1.3. Gene wide expression analysis
6.3. Evolution
7. Gene regulation
7.1. Types of gene regulation
7.1.1. Constitutive
7.1.2. Regulated
7.1.2.1. Long term
7.1.2.1.1. ex: human haemaglobin
7.1.2.2. Short term
7.1.2.2.1. ex: apoptotic genes
7.2. Transcriptional gene regulation by positive/negative control
7.2.1. Regulatory proteins for transcriptional gene regulation
7.2.1.1. Negative (repressor)
7.2.1.1.1. passive negative control
7.2.1.1.2. active negative control
7.2.1.2. Positive control (activator)
7.2.2. In Prokaryotic
7.2.2.1. Example: lac operon in E.coli
7.2.2.1.1. LacI repressor
7.2.2.1.2. CRP gene (CAP protein)
7.2.2.1.3. I* (initiator binding element)
7.2.2.1.4. Promoter (RNA polymerase binding site)
7.2.2.1.5. Operator (lacI repressor binding site)
7.2.2.1.6. lac Z
7.2.2.1.7. LacY
7.2.2.1.8. lacA
7.2.2.2. Example: cI repressor in lamba lysogeny
7.2.2.2.1. cI repressor
7.2.2.2.2. operator (cI binding site)
7.2.3. In Eukaryotic
7.2.3.1. Example: galactose utilization in yeast
7.2.3.1.1. GAL80 (repressor)
7.2.3.1.2. GAL4 (activator)
7.2.3.2. Example: in mamalian cells
7.2.3.2.1. negative control is called silencer
7.2.3.2.2. positive control is called enhancer
7.3. Transcriptional gene regulation by regulating chromatin
7.3.1. in prokaryotes ( none, no histone)
7.3.2. in eukaryotes
7.3.2.1. Structure and key features of chromatin
7.3.2.1.1. made of DNA wrapped around histone (octomer)
7.3.2.1.2. chromatin structure is variable
7.3.2.1.3. chromatin structure is dynamic
7.3.2.1.4. chromatin structure is heritable
7.4. Translational gene regulation
7.4.1. In prokaryotes
7.4.2. In eukaryotes
7.4.2.1. affecting global tranlational
7.4.2.1.1. 5 cap recognition inhibition by 4eBP
7.4.2.1.2. met-TRNA complex formation inhibition by GCN2
7.4.2.2. affecting gene specific translation
7.4.2.2.1. specific binding protein bind to 3' UTR of mRNA
7.4.2.3. post-translational modification
7.4.2.3.1. many example but notable is