immunology

• Innate immunity provides immediate protection against pathogens, while adaptive immunity develops over time and provides long-lasting memory responses.
• Recombinant DNA engineering provides the means to assemble genetic information in new combinations in a directed way, also known as 'genetic engineering'.
• Gene cloning, also known as 'molecular cloning', is central to recombinant DNA engineering technology.
• Gene of interest is linked to a vector that enables its amplification and propagation as a pure population of molecules in cells, usually bacterial.
• Molecular cloning of DNA is important for purifying and amplifying individual fragments of DNAs of genes of interest, obtaining their DNA sequences, determining the gene structure and regulation, performing site-directed mutagenesis to investigate function, expressing and purifying protein for biochemical/structural analysis, enabling genome analysis by creating overlapping clones of genomic DNA, and reintroducing genes into another organism, transgenesis, to acquire new functions and phenotypic changes.
• Type II restriction endonucleases cleave DNAs at specific bases.
• Type II methylases methylate DNAs at specific bases.
• DNA polymerases copy DNA from a primer 3’ end.
• Klenow DNA polymerse I is used to make a radioactive DNA probe from the entire DNA fragment using random hexanucleotide primers and a-32 P - dNTP.
• coli DNA polymerase I has 5’ to 3’ synthesis activity, 3’ - 5’ exonuclease activity (proof reading), and 5’ - 3’ exonuclease activity (primer removal).
• T7 DNA polymerase has very high processivity and is used for chain terminator DNA sequencing.
• Thermostable polymerases Taq, Vent, Pfu are used for the Polymerase Chain Reaction (PCR).
• T4 DNA polymerase has 5’ to 3’ synthesis activity and high level 3’ - 5’ exonuclease activity, making it useful for trimming restriction fragment ends.
• DNA polymerases can be used to radioactively label restriction fragments by using a-labelled dNTPs in the polymerisation reaction.
• Reverse transcriptase is used to make complementary DNA (cDNA) from RNA templates, initiating synthesis from an oligonucleotide primer.
• Klenow sub-fragment of DNA polymerase I lacks 5’ - 3’ exonuclease activity and is used for initiating DNA synthesis from oligonucleotide primers for radioactive labelling or chain terminator DNA sequencing.
• RNA polymerases make a RNA copy of DNA from a promoter.
• Reverse trancriptase makes a DNA copy of RNA from a primer 3’ end.
• DNA ligases covalently joins two DNA molecules or fragments.
• DNA ligase can efficiently join two DNA restriction fragments with compatible sticky-ends: Eco RI ends.
• DNA ‘sticky ends’ can associate with each other by hydrogen bonding between complementary bases and DNA ligase then restores the phosphodiester bond.
• The concentration of DNAs in ligations can be adjusted to give different outcomes: high concentrations result in inter-molecular ligation to produce linear concatenated DNA; low concentrations will result in intra-molecular ligation to produce circular DNA.
• DNA ligase requires a hydroxyl group at the free 3’ end and the phosphate group at the free 5’ end.
• Xho I requires a co-factor (rATP for T4 DNA ligase) that forms a covalent intermediate with the enzyme.
• Xho I functions in DNA replication to repair strand nicks and to join adjacent Okasaki DNA fragments.
• DNA ligase can also join restriction fragments with blunt-ends: Eco RV ends.
• Ligase can link two restriction fragments with compatible ends (e.g. vector DNA and desired insert) at low DNA concentrations to give a covalently closed circular product.
• Xho I (CTCGAG) catalyzes the formation of 5’ - 3’ phosphodiester bonds in double strand DNA molecules between juxtaposed 5’ phosphate and 3’ hydroxyl.
• This is an inter-molecular ligation, and a subsequent intra-molecular ligation.
• Enzymes used in recombinant DNA engineering include enzymes such as enzymes for DNA replication, enzymes for DNA modification, enzymes for DNA repair, and enzymes for DNA transcription.
• Restriction endonucleases and methylases were discovered from studying the host range of infectious bacteriophage.
• The structure of Bam HI restriction endonuclease and its binding to DNA is non-specific and then specifically as viewed on the left vertical to the helix, and as viewed on the right down the axis of the helix.
• 5' termini of each strand in the cleavage product(s) retain the phosphoryl group from the phosphodiester bond; the 3' termini are hydroxylated.
• CTTAAG GAATTC CTTAA G AATTC Eco RI cleavage of non-methylated DNA.
• CTTAAG GAATTC Eco RI methylase Eco RI CTTAAG GAATTC Fully methylated Hemimethylated No cleavage of methylated DNA.
• Bam HI restriction endonuclease binds as a homodimer and forms a 2-fold symmetric enzyme DNA complex.
• The enzyme slides along the DNA helix and alters conformation when the specific recognition sequence is encountered, resulting in tighter atomic interactions and cleavage of both strands.
• Methylase activity is on a separate protein.
• Type II restriction enzymes cleave DNA to produce three types of termini: 3’ recessed ends; blunt ends; 5’ recessed ends.
• In addition to the restriction enzyme cleavage being blocked by its cognate methylase, cutting can be blocked by other DNA methylation patterns.