Final exam - LM3 Flashcards
PCR steps
- add oligonucleotide primer
- denature
- anneal
- extend
E coli genome vs human genome
- e coli = 4.7m bp
- human = 3.2b bp
Sanger sequencing
- DNA from PCR combined with a primer
- DNA polymerase binds
- dNTPs bind
- ddNTP cause termination
- product put into capillary sequencer to detect tagged DNA products
If you want to sequence something you dont know
- clone unknown into a plasmid using universal sequencing primers
- ligate unknown DNA to known DNA as known DNA can be used as primers
Illumina sequencing
- ligate adaptors
- clusters form on flow cell
- bridging amplification via clonal PCR = add ddNTPs
- sequence via synthesis at each cluster
- scan surface for fluorescent ddNTPs
- short reads only
advantages of illumina
- accurate
- high throughput
Pacbio
- single molecule
- real time sequencing
- long reads
- expensive
- sequence by synthesis = ddNTPs
Nanopore
- single molecule
- very long reads
- portable
- fast and convenient
- charge measured for each base that passes through pore
How to assemble a new genome
- build a scaffold using long reads (pacbio or nanopore)
- overlay with short reads (illumina)
- collect reads and align for reference
challenges in assembling genomes
- repeat sequences which look identical
- telomere sequences are identical between chromosomes
- pseudogenes that are similar
- large deletions/insertions that vary between individuals
Mutations
changes made in DNA sequence
Variants
different versions of DNA sequences that differ by one or more mutations
Synonymous mutation
alter codon but same AA
missense mutation (conservative)
alter codon and changes AA that is chemically similar
missense mutation (nonconservative)
alter codon and different amino aci
nonsense mutation
premature stop codon
Exome sequencing
- used when most variants are exomic
- cheaper than full genome
- cant see large deletons
- exome is pulled out of genome after DNA shearing into ssDNA
- DNA is sequenced and mapped to genome
likelihood of mutation being casual determined by
- effect on coding sequence
- knowledge of function
- homology in other species
De novo mutation
neither parent will have the same variation as offspring
Why we want to clone
- need more DNA
- may want to express recombinant proteins to test function or for medicinal use
What happens if you reinfect same strain of E.coli
same titre of bacteriophage if you infect with a different strain you get a reduced titre as you have different restriction enzymes
Why bacteria cuts phage DNA but not its own
DNA is methylated VIA methylases which protects DNA
Restriction enzyme characteristics
- usually palindromic
- 4-8 nucleotides in length
- cleave phosphodiester bond at recognition site
2 types of restriction enzymes
- sticky cutter = cut at different points, higher in frequency
- blunt cutter = cut at same point
Cloning steps
- cleave using restriction enzyme
- ligate into plasmid vector
- clone
Ligation
- T4 DNA ligase reconnects phosphodiester bonds between 3-OH and 5-PO4
- needs ATP for energy and Mg as cofactor
- forms recombinant DNA plasmid
components of plasmid vectors
- polylinker = destination for DNA
- amp resistance gene for positive selection
- origin of replication
- lac Z = inactivated when DNA added
Inserted DNA into plasmid can be directional or non-directional
- directional = use 2 different cloning restriction enzymes, 5’-3’ direction known
- non directional = single RE used or blunt ended
How to transform plasmid into E coli
- chemically competent E.coli = treat cells with CaCl and heat shock making it porous to DNA
- electroporation = zap cells so they take DNA up
Implications for non directional inserts
additional screening required if orientation is important
what is screening
restriction digest DNA to identify correct clones and orientatione
end modifications in clone
- dephosphorylation = stops vector fro recircularise
- addition of single A = makes compatible for TA cloning
- blunting = makes compatible for blunt cloning
- phosphorylation = allows ligation
Why we might want more plasmid DNA
- construction of cDNA libraries
- subcloning small fragments for RE mapping
- to produce protein or RNA for vaccines
Expression plasmid vector features
- origin of replication
- ampicillin resistant gene
- promoter for highly expressed gene
- polylinker
- lacI gene for inducible expression
- HIS tah
- repressor of primer to control copy number
Problems with e coli recombinant expression vector
- proteins expressed in bacteria are often insoluble
- post translational modifications are different from mammals
How to optimise recombinant protein expression
- strong promoter
- inducible expression
- optimise translation using host codons and avoiding rare codons
- optimise stability by removing protease
- optimise solubility through fusions making it soluble
- post translational modifications
Factor VII production
- expressed via T7 RNA pol and T7 late promoter through a plasmid expression vector
- T7 RNA pol is expressed via Lac promoter in recombinant E.coli chromosome
- RNA pol activates promoter on vector to drive production
Insulin recombinant production
- insulin cleaved into A and B chains to improve solubility
- purify chains and then refold
- oxidise cysteines to form disulfide bonds to form active insulin
Genomic library
a set of clones containing all of the genomic DNA of an organism
cDNA library
a set of clones or a pool of cDNAs containing DNA copies of all mRNAs expressed in a certain cell type
Construction of a genomic library
- prepare DNA inserts with blunt cutter and gel purify large fragments
- insert DNA into BAC vector which has been cut with blunt cut RE
- transform and culture
Construction of cDNA library
- isolate and collect mRNA
- synthesise cDNA from mRNA via oligoT and reverse transcriptase
- insert cDNA into bacterial plasmid vector
- transform plasmids into bacteria and culture
- isolate plasmid and purify
- sequence DNA
What does cDNA show
shows tissue specific expression
what does genomic library show
intron and promoter info
problem with PCR
you have to know enough about your target gene as you have to design primters
reverse transcriptase PCR (cDNA)
- anneal oligo-dT primer to mRNA with poly A tail
- make cDNA VIA reverse transcriptase
- digest away RNA with enzyme and make a DNA copy of cDNA
- you can add restriction sites
- make DNA with primers and linker connecting primers 5’ end and amplify
- DSDNA made and can clone into vector
what happens if you dont add linkers to DNA copy of cDNA
you will have a DNA pool
Why sequence RNA
- characterise complexity of genes expressed in a particular tissue
- quantify expression levels of transcripts and compare samples
- can discover novel genes, exons and splice isoforms
- allows assembly of coding sequence or transcriptome
illumina with cDNA
make mRNA to cDNA via reverse transcriptase
Single cell RNA sequencing
- shows you the cells within a tissue
- input a single cell which can show expression variability and heterogeneity
advantages of single cell RNA over bulk
- can reveal new cell types
- can identify new cell types and genes involved in disease
- can reveal cells that are lost or damaged in disease process
- can be used to test targeted effects of therapies
How is polymorphism assesed
by measuring allele frequencies at individual loci
how are markers chosen
based on how polymorphic the gene is
restriction fragment length polymorphism
differences in DNA sequence at restriction sites recognised by restriction enzymes
what are restriction fragment length polymorphisms caused by
base changes that remove sites or add sites
Why have microsatellites and SNPs replaced RFLPS
they are based on PCR so they are faster,cheaper and automatable
Microsatellites
- small DNA sequence repeats
- interspersed frequently throughout genome
- highly polymorphic between individuals so is a good marker
- high frequency rate of mutation
- measured via fluorescence with sanger
SNPS
- single nucleotide variant
- if there is a higher number of base changes among individuals with a disease this is informative
- high throughput
- less polymorphic than microsatellites
Methods for sequencing markers
- affymetrix
- illumina SNP genotyping
Affymetrix
- DNA sheared and oligonucleotides are synthesised to glass slide
- fluorescently tag DNA
- detect fluorescence by laser
- a perfect match will have high amounts of fluorescence
- genotyping via hybridisation
illumina SNP genotyping
- genotyping via oligo extension
- isolate genomic DNA and shear
- anneal oligos adjacent to SNP
- fluorescently label oligonucleotide
- increased fluorescence when it anneals to SNP
applications of DNA markers from genome
used to construct fine resolution genetic maps and population and genetic diversity studies
applications of DNA markers linked to specific trait loci
identification of single genes affecting traits and multigenic trait analysis
Ways to put DNA into cells
- transformation
- projectile gun
- injection
- virus
Transformation
genetic alteration of a cell resulting from direct uptake and incorporation of exogenous genetic material from its surroundings through cell membrane
Transfection
process of deliberately introducing naked or purified nucleic acids into eukaryotic cells
Transduction
process by which foreign DNA is introduced into a cell by a virus or viral vector
Transgene
a gene or genetic material which has been transferred naturally or by genetic engineering from one organism to another
Genome editing
genetic engineering where DNA is inserted,deleted,modified or replaced in genome of a living organism
