Genomics Flashcards
1
Q
What are the steps of DNA isolation?
A
- Cell lysis
- DNA purification from the cell extract
- Concentrate DNA
- Measurement of DNA purity and concentration
2
Q
What kind of things do we not want in our DNA sample before PCR?
A
Protein
Ribosomes
mtDNA
Lipid
Plasmids
3
Q
What is a detergent?
A
Use enzymes to disrupt cell membranes.
4
Q
What kind of detergent is used on plants?
A
Cellulase.
5
Q
What kind of detergent is used in bacteria?
A
Lysozyme
6
Q
What kind of detergent is used in Eukaryotic Cells?
A
Sappanin
7
Q
How is osmotic pressure used during Cell Lysis?
A
Excess water moves into the cell when the cells are placed in a HYPOtonic solution, this causes the cell burst.
8
Q
How is freeze thaw used in cell lysis?
A
Repeated cycles of freezing and thawing ruptures cell membranes through ice crystal formation.
9
Q
What kinds of grinding techniques are used in cell lysis?
A
Pestle and mortar.
Bead mill.
Vortex with beads.
10
Q
How is DNA purified by phenol-chloroform extraction?
A
Lysed cells or tissue are mixed with equal volumes of a Phenol: Chloroform mixture.
They then form the interface due to the precipitated protein in the middle.
This is because water doesn’t mix with the Phenol:Chloroform mixture creating 2 distinct phases.
11
Q
How is DNA purified using commercial kits?
A
- Column contains a silica membrane that binds DNA in the presence of a high concentration of salt.
- Impurities such as salts are washed away.
*Then a low salt buffer such as water or 10 mM Tris·Cl, pH 8.5 is used to release the DNA from the membrane and collect it.
12
Q
What are restriction endonucleases?
A
Molecular scissors that cut DNA in precise locations.
Enzymes produced by bacteria to protect against viral DNA infection
Restriction Enzymes cut the foreign DNA
13
Q
Why are restriction endonucleases used in the laboratory?
A
- To make recombinant DNA molecules (cloning)
- To cut DNA into defined fragments (DNA fingerprinting and mutation analysis)
14
Q
How do restriction enzymes cut DNA?
A
*Make one cut in each of the sugar phosphate backbones of the double helix (breaks bond between 3’O and P) at their recognition site in the presence of Mg2+
*Hydrolyses the phosphate group
*Cut ends have a 5’ phosphate
15
Q
What are the Principle of agarose gel electrophoresis?
A
*Polymerised agarose is porous, allowing for the movement of DNA (acts as a “molecular sieve”).
*Samples enter the gel and migrate according to charge, size and shape
*Visualise with intercalating dyes (we used Nancy Red- safe)
16
Q
Which molecules move faster in agarose gel
electrophoresis?
A
Smaller/compact molecules move more easily through gel than larger/long molecules
17
Q
How do we Determine the size of the DNA fragments after agarose gel electrophoresis?
A
- Compare size of product of interest with the DNA ladder.
- Look by eye and ‘guess’
- Plot a graph log10 and mm band migrated down the gel (use MW ladder to create a standard curve)
- Determine size of your products
- Graph of log DNA size of the markers by distance migrated creating a straight line
- Can then plot the distance the product of interest has travelled on the line and estimate the DNA size
18
Q
What is genome editing?
A
Genome editing is a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using nucleases
19
Q
Why do we need genome editing?
A
- enabling specific targeting of sequences within the genome without impacting the rest of the genome sequence
- Potential to cure genetic diseases in a patient specific manner
20
Q
What is CRISPR/Cas9?
A
Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) and CRISPR Associated (Cas) proteins
21
Q
What are the component systems that operate as complexes within CRISPR/Cas9?
A
- Cas9 - Protein component
- crRNA – RNA component
- tracrRNA – RNA component
22
Q
What is the main function of CRISPR/Cas9?
A
A Adaptive Immune Regulator in prokaryotes.
23
Q
How does CRISPR act as an adaptive immune regulator in initial infection ?
A
- Invading DNA recognised and cut by Cas1-Cas2 protein complexes into fragments termed protospacers
- Protospacers integrated into CRISPR locus located in the bacterial genome
24
Q
How does CRISPR as an adaptive regulator in reinfection?
A
1.Upon viral reinfection, transcription of the protospacers to RNA is activated which bind to Cas9
2.Cas9/RNA duplex is recruited to complementary sequence on the invading strand of DNA
3. Cas9 cuts DNA strands creating a double strand
break to prevent infection
25
What is the structure of CRISPR locus?
* Transactivating RNA
* Operon of cas genes encoding Cas protein components
* Identical repeat array
* Spacer of invading DNA
26
What is the Cas operon?
*Cas operon encodes Cas proteins
required for DNA cleavage
*Distinct ‘types’ of Cas proteins
27
What are Protospacer Adjacent Motifs (PAM)?
*Protospacer Adjacent Motifs (PAM) enable Cas9-
mediated DNA cleavage
* Provides a mechanism to distinguish bacterial self DNA from non-self DNA
* Prevents bacterial CRISPR locus being targeted by Cas proteins
28
How does PAM Prevents bacterial CRISPR locus being targeted by Cas proteins?
* Cas9 will not cut invading DNA without a PAM site irrespective of Cas/gRNA binding – distinct sequences for each unique Cas protein
* PAM sequences are not present in the CRISPR locus
29
What is the structure of Protospacer Adjacent Motifs (PAM)?
2-8 base pair sequence 3-4 base pairs downstream of the cut site
30
What is gRNA made up of?
* tracrRNA
* crRNA
31
How is Engineered CRISPR/Cas used in studies?
* Deposition of the Cas9/gRNA complex at a desired locus of the genome will enable site-specific cleavage through nuclease activity
* The repair of the DNA break by endogenous DNA repair pathways enables specific genomic edits to be introduced
32
How is CRISPR/Cas9 used as a molecular tool in eukaryotic cells?
Overexpression of Cas9 and gRNA in cells allows site directed editing
33
What are the important characteristics of gRNA?
*The gRNA should contain a proto-spacer sequence (target sequence) upstream of the PAM site
*gRNAs should be selective to a single genome locus to avoid off target effects
34
What Cellular DNA repair pathways are critical for desired CRISPR-mediated DNA editing?
1. Homology-directed repair (HDR)
2. Non-homologous end-joining (NHEJ)
35
Why is DNA repair by Non-Homologous End Joining (NHEJ) error prone?
- introduces insertions or deletions (indels) into DNA
- Impacts gene function
36
Why is NHEJ important to CRISPR genome editing?
Key to CRISPR Knockout studies
37
Why is HDR important to CRISPR genome editing?
Key to CRISPR Knock-in studies
38
How does CRISPR-mediated gene knock out via NHEJ work?
* Target Cas9:gRNA complex to gene of interest
* DSB introduced
* Cell repairs the break via NHEJ
* Indels introduced generating a frameshift
* Premature stop codons introduced
* Normal gene product not expressed
39
How does CRISPR-mediated gene knock-in via HDR work?
* DSB introduced by Cas9:gRNA complex
* Introduce a template that the cell will use to repair the DSB through HDR
* PAM sites are removed from HR template to prevent re-targeting of region
* Inserts of several kilobases are possible
40
How is CRISPR genome editing ex-vivo?
1. Remove cells from the patient / donor
2. Edit genome
3. Screen / expand cell populations
4. Engraft cells back into patient
41
How is CRISPR genome editing in-vivo?
1. Package CRISPR/Cas in a delivery vehicle.
2. Deliver to patient.
42
How successful was CRISPR editing of CCR5 in vivo confers HIV-1 resistance?
* Long term CCR5 disruption was observed
* CCR5 disrupted HSCs were able to reconstitute a functional immune system
* Viral titre reduction and increase in CD4+ T cells demonstrated HIV resistance
43
How is an organisms genomic sequence obtained?
1) Obtain the organisms genomic DNA
2) Break the DNA into small fragments
3) Obtain the DNA sequence from all the fragments
4) Search for overlaps of identity between the DNA sequences of the different fragments to “reconstruct” the genome sequence
5) Fill in any missing “gaps” in the genome sequence
44
What is a Model Organisms?
Small genome – “value for money”
Easy organisms to manipulate
Provide information on fundamental biological processes
Technology development
45
How big is the human genome?
30000 - 40000 genes
46
What are the microarrays?
A set of DNA sequences representing the entire set of genes of an organism, arranged in a grid pattern for use in genetic testing.
47
What are the stages of conducting microarrays?
Exact RNA is put through reverse transcription.
This becomes cDNA and is then put through in vivo transcription.
This leads to biotin labelled cRNA which is put random fragmentation.
Fragment biotin labelled cRNA is hybridised to gene chips.
We then wash away non-specific binder and stain with SP.
This array is the scanned with a laser, detect florescence with CCD, read image into computer.
48
What is High-throughput Sequencing?
*Sometimes called ‘next generation sequencing’
*Refers to range of technologies
49
What is illumia sequencing?
*Fragments of DNA bound to solid surface (flow cell)
* Binding enabled by special sequences ligated to fragments
*Solid-phase bridge PCR forms clonal clusters
*Sequencing proceeds in cycles
*Modified nucleotides with fluorescent group which blocks extension
*Reversible termination allows sequencing to proceed to next cycle
50
What is RNA sequencing?
*Use of high-throughput sequencing technologies to get information about a sample’s RNA content
*mRNA (and other RNAs) are converted to cDNA
*cDNA used for sequencing library generation
*Allows quantification, profiling and discovery of RNA
51
What are the steps to illumina sequencing?
1.Construction of a library for next generation techniques (NGS) which involves the addition of specific adapters to the DNA or cDNA fragments to be sequenced.
2. Amplification of library fragments.
3. Sequencing of the fragments through cycles of biochemical reactions. During the reaction cycles, information is acquired which allows, by specific software, to reconstruct a DNA or cDNA sequence.
52
What are the stages of RNA sequencing?
1. RNA extraction
2. Reverse transcription into cDNA
3. Adapted ligation
4. Amplification
5. Sequencing
53
What needs to be considered when running RNA sequence?
*Big data sets require expert processing
*Expression data can be noisy
* Careful experimental design important
*Easy for confounding factors to dominate
*Good practise same as for any statistical approach
54
What are the stages to ChIP-Seq?
*Cross-link proteins to DNA
*Isolate DNA and shear
*Immunoprecipitate protein of interest
*Reverse cross-linking
*Purify DNA
*Sequence
55
What is ATAC-Seq?
Assay for Transposase-Accessible Chromatin
56
What are the stages of ATAC-Seq?
*Relies on transposase Tn5
* High activity transposase
* Highly efficient cutting of exposed DNA
* Ligation of adapters to ends
*Adapter ligated fragments isolated, amplified and sequenced
57
What is Bisulphite Sequencing?
*Bisulphite treatment is used to determine the methylation state of DNA
*Methylated cytosine protected from deamination
*Unmethylated cytosine converted to thymine (via uracil)
58
Why are mice a good model orgainsim?
*We share ~95% of our genes with mice.
*Mice have a short life cycle
59
Why are mice difficult model organisms?
Relatively expensive to keep.
60
What licenses are required to use mice models?
‒ Personal license for the researcher
‒ Project license for the study
‒ Establishment licence for the place where the study is carried out
61
What are in vitro models?
*Different cell types interact with each other constantly within living tissues.
*These interactions are impossible to model (at this point) outside of animals.
62
What kinds of mouse models do we use to study human development and disease?
1. Transgenic mice
2. Knock-out mice
3. Knock-in mice
63
What is the Standard Transgenic Approach?
1. DNA (gene of interest) is microinjected into the pro-nucleus of a fertilized mouse oocyte
2. Injected oocytes are transferred to a pseudo-pregnant recipient mouse
3. ALL offspring are screened for expression of the transgene by DNA analysis
64
What is the Gene-targeted transgenic approach?
1. A isogenic transgene with a drug selection gene is introduced into embryonic stem (ES) cells
2, Drug selection is used and surviving cells are screened for the correct integration of transgene
3. Correctly targeted cells are micro-injected in mouse blastocysts
4. Blastocysts are transferred to pseudo-pregnant recipient mouse
5. Chimaeric offspring are identified and mated to test for germline transmission of transgene
65
What is the structure of the transgenic approach vector?
*gene of interest
*relevant promoter
*3’ protein tag for detection
*poly A tail
66
What are the strengths of transgenic mouse models?
*Cheap & cheerful – relatively easy to make
*Multiple founders are generated
67
What is the weaknesses of transgenic mouse?
*Can not control site of integration into genome
*Wild type gene product is still present
68
What is gene trapping?
Gene trapping is a high-throughput approach that is used to introduce insertion mutations across the genome in mouse embryonic stem cells.
69
What happens when you insert the gene trap?
*disrupts gene function
*reports gene expression
*provides a convenient tag for the identification of the insertion site
70
What is Cre recombinase?
A tyrosine recombinase enzyme derived from the P1 Bacteriophage and catalyses site-specific recombination between two LoxP sites
71
What is flippase?
A tyrosine recombinase enzyme derived from the the baker's yeast - Saccharomyces cerevisiae - and catalyses site-specific recombination between two FRT sites
72
What is the The cre-lox system for conditional alleles?
1. Following manipulation the mouse has a targeted “floxed allele”.
2. A second mouse is transgenic for cre recombinase expressed under the control of a promoter.
3. The two mice are crossed together to generate a mouse line that carries both alleles: “floxed” and “cre”.
4. Result is the tissue specific deletion of the “floxed allele” ONLY in tissues where cre recombinase is expressed.
73
What are the strengths of mouse models?
‘Genetraps’ available for virtually all genes from
74
What are the weaknesses of knockout mouse models?
May not accurately model a known human disease
75
What is knock-in technology?
Knock-in technology is mostly used to introduce a human mutation into the mouse genome
76
What are the weaknesses of knock-in models?
Traditionally time consuming (18m) and expensive (£20K)
Remaining LoxP site may be a problem
77
What are the strengths of knock-in models?
‘Genomically relevant’ mouse model of a human disease
78
What is a transgenic mouse?
Currently, the phrase "transgenic mouse" generally refers to any mouse whose genome contains an inserted piece of DNA, originating from the mouse genome or from the genome of another species.
