BBL WEEK 1

-Prokaryotes: Bacteria + Archaea - lack nucleus, simple cells, v small

-Eukaryotes: Animals + Plants - mbo with specialised functions, complex, larger

COMMON FEATURES OF CELLS:

• Surrounded by lipid membrane
• Respond to environment
• Contain DNA in the form of genes, carrying instructions for building proteins
• Common building blocks: a.a, proteins, carbohydrates, lipids, nucleic acids
• Proteins involved in structural support, communication, repair, catalayse reactions, recycling, making new organelles and cells etc
• More complex cells-specialised mbo
• highly dynamic, constantly changing, compartmentalised- faster, if diffusion occured in large volumes it would limit speed of reactions.

P ~ 1 micrometer³

E ~ 1000 micrometers³

VIRUSES:

• e.g bacteriophage
• much smaller than P such as E.coli
• Too small to function on their own-need host

COMPARTMENTALISATION:

• Cells change number + type of organelles- allow cell to take on different functions so specialise.- creating organs etc.
• Different combinations of s cells creates diversity of multicellular life.

COMMON ANCESTRY:

• Categorisation- linnaeus 1707-78- systema narturae Animal, plant, mineral kingdoms- discoveres 6000 plants, 4200 animals, nested heirarchy: class, order, genus, species
• LUCA-evidence for LUCA is biochemical, genetic, cytological, taxonomic, biogeographical, paleontological, gene duplications lead to differentiation.

MICROSCOPY:

Light:

• lenses magnify light
• light focused using collector lens onto mirror, bounces light through condenser lens onto specimen
• light from specimen is magnified by objective + projection lenses until it arrives at detector, limit 200nm small 

Electron:

• e- instead of light, lenses are electromagnetic
• see smaller objectus due to smaller wavelength of e-
• can't produce colour images or view live cells as they need a vacuum

Fluorescence:(light microscopy)

• part of light spectrum- relies on ability of fluophores to emit light when stimulated.
• use photons, emit photons of light with a higher wavelengthh, producing a different colour. Can be used to identify antibodies which bind to photons.

GREEN FLUORESCENT PROTEIN:

• Optimised for sea temp, existed as a tetramer
• weak fluorescence, easily bleached, unstable
• mutants created-folded correctly and worked efficiently at 37`c, by changing phenylalanine 64 to leucine
• created tri-peptide sequence SER65-TYR66-GLY67 changing serine64 to threonine dramatically increased fluorescence and photostability and shifts emission to green spectrum
• changing tyrosine 66 to tryptophen caused a shift to blue
• insert GFP before stop/start codon
• super-resolution lm allow cells to be viewed almost like an e-m, absorbs blue, emits green

CELL CULTURE:

• need cell supply
• cells from individuals/animal tissues vary as they are genetically diverse so lack consistency
• cells removed from natural environment can get stresses and sdie without proper nutrients
• isolated tissues can be kept functional for several days in simple salt solutions, individual cells are harder to culture

HAYFLICK LIMIT:

• most Ecells have limited lifespan and stop proliferating around 40-60 cell divisions even with nutrients
  
• Telomere shortening limits no. cell divisions-tumour cells are effectively immortal as they rebuild telomeres using telomerase

HeLa CELLS:

• Isolated from Henrietta Lacks 19522 cervical tumour, some cells isolated
• 1st human cell line-used in models,used to test polio vaccine 1950s, contaminate other cultures

PHE, ATCC:

• Worldwide repositories for cell lines
• Typical culture media: Salts(CaCl2,KCl,NaHCO3)A.A(17 essential)Vitamins(Folic acid, riboflavin)glucose, phenol red dye
• Adherent-need to attach to surface- when cells run out of space, they split by trypsin(enzyme)
• passagine-1-2 times a week
• some cells grow in suspension

• aseptic technique
• use of antibiotics + laminar flow cabinets maintain sterile conditions with incubators 37`c

HISTORY OF GENETICS:PRE 20TH CENTURY:

• Aristotle-animals look same by magic-environment has huge effect on organism, believed in blending(removed variation)
• Gregor Mendel-fryer0peas-cross counted yellow and green peas
• August Weismann-mouse tails
• chromosome theory-correct number of chromosomes

MODERN GENETICS:

• Bateson + Punnet 1905, saunders, independent assortment, linkage- 2 characteristics from parents pass down
• TH Morgan + genetic mapping-alfred henty sturteu and nelty stevens-fruit flies, mapping
• Biometry-e.g pedigree diagram
• multiple non blending genes
• Molecular biology-mutagenesis(muller/auerbach), x rays, chemicals

GENETIC MANIPULATION:Take piece of DNA from one place, put it somewhere else(cloning)

1-purify DNA in sufficient amounts from cells/tissues/organisms

• extract cells via lysis or mechanical disruption
• freeze and crush
• use detergent
• shake with beads

2-manipulate DNA in lab

• restriction endonuclease-some strains of bacteria were more resistant to viral infections than others
• bacteriophages are host restricted
• recognition sites for endonuclease
• cut restriction sites that are specific, creating sticky ends or blunt end fragments.
• DNA ligase rejoins DNA molecules so new phosphodiester bonds form using ATP or ADP+
• quality control using gel electrophoresis-agarose gel separates mixture of nucleic acids based on size:-ve move toward +ve electrode vice versa, and shorter fragments move faster so are further along.
• piet borst(ethidium bromide)

3-provide large amounts of DNA to study

• Natural replication:plasmid/vector delivery of 2 DNA into host cell for amplification-plasmid vectors are modified forms of circular extrachromosomal DNA in bacteria-restricter sites and marker genes
• Landa phage vectors-carry large DNA sequences than plasmid vectors
• Cosmids-packed into phage body, use plasmid genes to direct replication within host
• Expression vectors-regulatory sequences for transcription and translation. Makes as many copies of protein coded by gene as possible
• In vitro replication: PCR

Self replicating DNA

Selectable markers and restriction enzyme recognition sites(unique)

CLONING:

• process used to create exact genetic copies of DNA fragments, cells and organisms

GENOMIC LIBRARY:

• look at gene structure and compare genomes between organisms

cDNA library:

• Identify genes encoding proteins in a specific tissue

DNA PROBES:

• Hybridization probe-fragment of DNA/RNA of variable length (usually 100-1000 bases long) which can be radioactively labelled
• used in DNA/RNA sample to detect presence of nucleotide sequences (DNA target) cbp to the probe

HYBRIDIZATION:

• DNA denatured, so single stranded at 60`c, high salt
• Stringency-conditions increase, probe doesn't bind to target. Decrease, probe binds to unrelated targets
• Rate depends on DNA conc and time and GC content and length of DNA

HYBRIDIZATION USED IN:

• Southern blotting-DNA digested by restriction enzyme and separated on gel-detect specific DNA sequence in complex mixture e.g genomic DNA. Cut restriction enzymes, run on agarose electrophoresis gel, transfer DNA from gel to nitrocellulose membrane. Fix DNA to membrane permanently with UV/heat, hybridise membrane to radioactive probe, detect specific bands with autoradiography, study genomic sequence
• Northern blotting-RNA instead of DNA used to look at gene expression in tissue/size of transcripts
• In situ hybridization-probe tissue to see where gene is expressed-identify and characterise numerical and structural chromosome abnormalities, detect microscopial invisible deletions, detect sub-telomeric aberrations, prenatal diagnosis of common aneuplodies 
• Same as S/N blotting but used on tissue to see where mRNA is expressed
• Colony hybridization-edtection of clones-bacterial colonies containing recombinant DNA are grown and blotted to nitrocellulose. Can then hybridise a probe same as S blotting. Colonies on agar plates stay active, and once correct colony has been detected, can be picked and grown up for further work

• Microarrays-use principles of cbp and hybridization to identify expression of a gene in particular cell/tissue, used to identify specific point mutations (SNPs) in sequence of DNA.
• PCR-specific DNA fragment amplification based on DNA replication catalysed by DNA polymerase. PCR uses ability of DNA polymerase to synthesize new strand cbp to template strand. Primer is needed as DNA polymerase can add a nucleotide only onto pre existing 3` OH group. DNA polymerase then elongates primer's 3` end by adding more nucleotides to generate an extended region of double strand

Natural replication:Plasmid/vector delivery of DNA into host for amplification

PCR:

• Kary Mullis 1985
• amplifies small pieces of DNA into millions of copies
• creates workable quantities for research, useful when DNA is degraded
• Each cycle takes a few mins, so lots are made in jut hours
• some details of sequence must be known in advance
• sensitive to small amounts of conamination

PCR CONTINUED:

• chain reaction
• exponentially grow population of identical DNA molecules
• Denaturation-separate 2 nucleotide strands of DNA
• Primer annealing-primers bind to ** DNA
• Extension-nucleotides added to primers 5` to 3` to form ds copy of target DNA
• Detects and sequences v small amounts of DNA quickly
• modify DNA by changing primers
• used in paternity, SNP, predict future disease, detect rare species in aq environment (cDNA)

DNA SEQUENCING:

• Uses concept of DNA replication
• DNA polymerase drive the DNA sequencing by synthesis technologies:
• Sanger sequencing-chain termination or dideoxy sequencing
• next generation sequencing
• watson and crick 1953 (1956-23 pairs of chromosomes)(1977 fred sanger invented process)

ENZYMATIC DIDEOXY DNA SEQUENCING-SANGER:

• REQ:
• many identical copises of DNA to be sequenced
• Oligonucleotide primer cbp to short stretch of DNA
• DNA polymerase
• Deoxynucleotides (dNTPs)
• Dideoxynucleotides (chain terminating nucleotides)

 1)DNA mixed with primer in excess nucleotides, terminator bases with fluorescent markers attached                                                                                                                                                                                                                                   2)mixture placed in thermal cycler 96`c DNA denatures, 50`c primers anneal, 60`c polymerase builds new strands                                                                                                                                                                                                     3)Whenever terminator base is added, chain stops to produce different fragments                                                 4)Fragments are separated by length by capillary sequencing similar to GE                                                               5)Fluorescent markers used to identify final bases and lasers detect diff colours                                                             6)order of bases in capillary tubes shows sequence of complimentary strand                                                                     7)info is fed into computer and sequence can be determined 

NEXT GENERATION SEQUENCING:

• uses single template strand immobilized and amplified to produce an enormous number of identical fratgments
• thousands or hundreds of thousands of fragments (400-1000 nucleotides long) are sequenced in parallel
• type of high throughput technology