CMB2000 - research skills Flashcards
Principally, what does PCR do?
Amplify DNA
DNA replication in prokaryotes
- DNA helicase unwinds DNA -> replication fork
- RNA primers bind template -> dsDNA
- polymerase joins nucleotides together
what happens on the 5’->3’ strand during DNA replication (DNA polymerase)
DNA pol can only run 3’->5’
leads to okasaki fragments on lagging strand
joined together later using ligase
role of sliding clamps
keeps polymerase in place on the DNA during eukaryotic DNA replication
Components needed for PCR
DNA template, Primers, Taq polymerase, dNTPs, MgCl2 (co-factor), 10x tris HCl buffer
Steps of PCR
- dsDNA is boiled at 95C -> ssDNA
- cooled to 55C to anneal primers
- heat back up to 72C, extend with polymerase and dNTPs
- repeat to amplify DNA between primer locations
How are PCR products detected?
stained with an intercalating gel (e.g. ethidium bromide - red under UV), and products are ran on agarose gel using electrophoerisis
How to do PCR if you start with RNA?
reverse transcriptase PCR converts RNA to cDNA first
SYBR green
fluorescent marker that binds to grooves of dsDNA
TAQMan
fluorescent, uses probes with reporter and quencher
more PCR product = more fluorescence
reference genes
housekeeping genes at a constant level of expression
Why is PCR clinically valuable?
Sensitive, specific, cheap, rapid, robust
3 uses of PCR in diagnosis/prognosis
- genotyping the patient
- genotyping the pathogen
- phenotyping the disease (Snapshot in time)
what is PCR-RFLP?
PCR Restriction fragment polymorphism
- uses restriction endonucleases
what is ARMS-PCR?
Amplification refractory mutation system
- detects allelic variants using allele specific primers
three conventional diagnostic techniques that can be replaced with PCR
microscopy, culture, patient antibody response
key tehnique for phenotyping a disease?
quantitative PCR
what is the ct value?
cycle threshold - how many cycles needed before fluorescence is visible.
lower ct = more nucleic acid/DNA
at what stage would you expect the lowest ct value?
immediately after infection, before immune system has had time to act
4 steps of DNA isolation
- cell lysis
- DNA purification from cell extraction
- concentrate DNA
- measure DNA purity and concentration
methods of cell lysis/DNA extraction
Biological - enzymes (lysozyme in bacteria, sappanin in eukaryotic)
physical - osmotic pressure/ freeze-thaw
mechanical - grinding, friction, shearing
DNA purification
Phenol-chloroform extraction
- then centrifuged
OR
column purification commercial kits
- silica membrane
Restriction endonucleases
molecular scissors that cut DNA in precise location
what are restriction endonucleases used for?
- make recombinant DNA molecules
- cut DNA insto defined fragments
Types of REs
sticky ends or blunt ends
RE recognition sites are often….
palindromic
Agarose gel electrophoreisis
separates DNA fragments
agarose = polysaccharide from seaweed
principle of electrophoreisis
- polymerised agarose is porus -> DNA can move along it
- samples migrate along the gel according to size/shape/charge
- smaller/compact molecules will move further
- visualise with intercalating dyes
what does CRISPR/Cas9 stand for?
CRISPR = Clustered Regulatory Interspaced Short Palindromic Repeats
Cas = CRISPR associated proteins
3 components of CRISPR/Cas9 complex
- Cas9
- crRNA
- tracrRNA
CRISPR as an adaptive immune regulator
- invading DNA recognised and cut by Cas1-Cas2 -> fragments
- protospacers integrated into CRISPR locus
- viral reinfection -> transcription of protospacers -> bind Cas9
- Cas9/RNA duplex recruited to invading DNA strand
- Cas9 cuts DNA stands -> DSB -> prevents infection
What does the Cas operon encode?
cas proteins required for DNA cleavage
components of the CRISPR locus
transactivating RNA
Cas operons
identical repeat array
spacer of invading RNA (between tracrRNA and crRNA)
Engineered CRISPR/Cas9 in the lab
- composite gRNA made using linker loop
- deposition of Cas9/gRNA at desired locus -> site-specific cleavage via nuclease activity
- repair of DNA break by endogenous DNA repair pathways allows specific genome edits
gRNA =
tracrRNA + crRNA
two mechanisms of cellular DNA repair machinery
- Homology directed repair (HDR)
- Non-homologous end-joining (NHEJ)
CRISPR-mediated gene knockout via NHEJ
Target Cas9-gRNA complex to gene of interest
DSB introduced
Cell repairs break via NHEJ (error prone)
Indels introduced -> frameshift -> premature stop-codons introduced
normal gene product not expressed
CRISPR-mediated gene knockout via HDR
DSB introduced by Cas9-gRNA complex
Introduce a template that will be used to repair DSB by HDR
PAM sites are removed from HR template -> prevents re-targeting of the region
Inserts of several kb are possible
PAM site
prospacer adjacent motifs
Ex-vivo delivery of CRISPR in the clinic
- remove cells from the patient/donor
- edit genome
- screen and expand cell populations
- engraft cells back into patient
in-vivo delivery of CRISPR in the clinic
- package CRISPR/Cas in a delivery vehicle
- Deliver to patient
Steps to obtaining genomic sequences from an organism
- obtain organism’s genomic DNA
- break DNA into small fragments
- Obtain the DNA sequence from all the fragments
- search for overlaps to help reconstruct the sequence
- fill in the missing gaps in the genome sequence
computer analysis of protein sequence
- predict function - role of model organism
- prediction of protein localisation
- prediciton of protein domains/modification
What does BLAST search do?
identify conserved domains
what does NetPhos search for?
potential serine/threonine/tyrosine phosphorylation sites
uses of genome sequence within an organism
identification of regulatory sequences
characterisation of protein families
regulatory sequences
identify all promotors containing a transcription factor binding binding site
Functional genomics experiments…
describe gene functions and interactions
microarrays measure…
hybridisation
illumina sequencing
fragments of DNA (library) bound to solid surface (flow cell)
solid phase PCR forms clonal clusters
only one base can be added per cycle - modified nucleotides with fluorescent group that blocks extension
reversible termination allows sequencing to proceed to next cycle
steps of RNA-Seq
poly A selection -> fragmentation
Random priming (to remove bias)
cDNA synthesis (RT)
End repair, phosphorylation and A-tailing
Adapter ligation, PCR amplification and sequencing
general experimental schema in sequencing
- Enrich
- Sequence
- Analyse
ChIP-Seq
Cross link proteins to DNA
Isolate and shear DNA
Immunoprecipitate protein of interest
reverse cross linking
purify DNA
sequence
ATAC-Seq
Assay for Transposase-Accessible Chromatin
Relies on transposase Tn5 (highly active, efficiently cuts DNA and ligase adapters to ends)
Adapter ligated fragments isolated -> amplified -> sequenced
bisulphite sequencing
Determines the methylation state of DNA
Methylated cytosine is protected from deamination
Unmethylated cytosine converted to thymine via uracil
BS-Seq
Identifies sites of methylation - sequences bisulphite treated and untreated samples
quantitative estimates of methylation
identifies hypo- and hyper- (transcriptional silencing) methylated regions
3 licences required for research on animals
- personal licence for the researcher
- project licence for the study
- establishment liscence for where study takes place
standard transgenic mouse approach
- gene of interest injected into pro-nucleus of fertilised mouse egg
- injected eggs transferred to pseudo-pregnant recipient mouse
- all offspring are screened for expression of transgene by DNA analysis
gene-targeted transgenic approach
- isogenic transgene with drug selection gene introduced to embryonic stem cell
- drug selection used- surviving cells screened for transgene integration
- correctly targeted cells are injected into mouse blastocytes
- blastocytes transferred to psuedo-pregnant mouse
5, chimaeric offspring are identified and bred -> germline transmission of transgene
transgenic mouse vector construct
tissue specific promoter - gene of interest - 3’ protein tag for detection, polyA tail
Cre recombinase
catalyses site-specific recombination between two LoxP sites
Flippase
catalyses site-specific recombination between two FRT sites
which gene allows straight forward knockouts
loxP
genes that allow conditional knock outs
FRT and LoxP
cre-lox system for conditional alleles
- manipulation -> targeted, floxed allele in mouse
- second mouse is transgenic for cre recombinase
- mice are crossed -> mouse with cre and floxed alleles
- tissue specific deletion of the floxed allele in tissues w/ cre recombinase
- can also have inducible cre expression
knock-in mouse
mostly used to introduce a human mutation into the mouse
uses CRISPR/Cas9
