1
Q
Purpose of detecting and quantifying DNA,RNA, and proteins
A
- Investigate their function in normal and diseased states
- Determine if certain genes are present in similar species
2
Q
Purpose of blotting
A
Detect SPECIFIC molecules within a mix of many different molecules (DNA, RNA, protein)
3
Q
Steps of Blotting
A
- Gel electrophoresis
- Transfer molecules to membrane paper
- incubate membrane with probe solution (hybridization)
- Autoradiography (X ray)
4
Q
In Situ Hybridization (ISH)
A
In vivo version of blotting for RNA and DNA
Radioactive probes
5
Q
fluorescence in situ hybridization
A
uses fluorescently labeled probes instead of radioactive probes
6
Q
Immunofluroescence
A
In vivo version of blotting for proteins
uses antibody probes and a fluorescence microscope
7
Q
Traits of restriction enzymes and sites
A
- Endonucleases
- Cleave phosphodiester bonds
- sites are palindromic (both strands have same sequence in antiparallel fashion)
8
Q
PCR purpose
A
Make billions of copies of fragment of dna
9
Q
PCR steps
A
- Pair of primers (20 nuc each) designed to base pair to end of target gene
- Added to solution with template, dATP/dGTP etc. and Taq
- Heated to denature, cooled to aneal, heated to synthesize
10
Q
Real Time PCR
A
Purpose: calculate amount of DNA in a sample at a given time
Measure intensity of fluorescent signal generated by dye
Calculation: X=2^(CT1-CT2)=2nd sample has X fold less DNA than 1st sample
where CT is the cycle threshold (number of cycles for signal to be detected above ground)
Used to compare relative ampunt sof diff DNA in sample
11
Q
Example of purpose of qPCR
A
1.diagnose cancer due to a somatic mutation - determine fraction of cells in tumour sample that contain mutant gene vs WT
- compare relative amount of same dna ( rate of progression of viral infection in blood samples taken at different times)
12
Q
PCR for RNA
A
- Purify mRNA from tissue or specific cells
- Incubate with reverse transciptase, dNTPs and oligo-dT primner (anneals to polyA tail)
- Convert to cDNA (double stranded version of mRNA molecule)
Use cDNA as you would DNA in same tests
13
Q
multiple cloning site/polylinker
A
Restriction enzyme site that does not repeat in a plasmid - plasmid only cuts at one location
14
Q
Plasmid Vectors
A
- Plasmid Vectors: replicated independently of whole chromosome due to ori site.
- Good for drug resistance testing and determine which bacteria contain resistant genomes
PRACTICAL: dna inserts disrupt gene lacZ in plasmid. it encodes enzyme B-galactosidase - cleaves compound X-gal when added to culture plate and turns blue.
THEREFORE: with insert - white. without - blue
SMALL INSERTS
15
Q
Expression Plasmids
A
- contain sequence that controls transcription and translation of insert dna
- can drive transcription constitutively (constant) or inducible (when signaled)
PRACTICAL:
1. pET plasmid 1 have T7 promoter - drives transcription of inserted gene in E.coli - expresses phage T7 RNA polymerase
- pET plasmid 2 uses lac operon components to inducible express gene in e.coli - lac operator site near T7 promter, contains lac1 gene - encodes lac repressor protein - binds lac operator - represses transcription. When IPTG added to growth media, lac repressor inactivated and T7 transcribes
- pET have epitope tag (His-tag). Epitope tags : purify recombinant proteins. short protein sequences - translated in frame at N or C terminus of recomb protein. Purified by affinity chromatography from E.coli extract - solution of e.coli cells broken open release recomb protein and e.coli protein - mixed with charged beads - only recomb bind to beads - tags bind to beads with recomb - beads washed - only recomb beads left - released by adding competing chemical. USED TO SYNTHESIZE AND PURIFY HUMAN INSULIN PROTEIN
16
Q
BACTERIOPHAGE VECTORS
A
- can hold different sized of inserts (double stranded)
- central part of genome removed with RE and replaced with insert
- Up to 15kb
- can be introduced to cells via phage
MEDIUM SIZE INSERTS
17
Q
Vectors for large inserts
A
- Fosmids
- Bacterial artifical chromosomes (BACs)
18
Q
Fosmids
A
- 35-45 kb inserts
-fosmid packaged into A phage particles - particles introduce insert into recipient e.coli cells
- cos sites (cohesive) : 12 bp sticky ends - turn linear phage dna into circular
- fosmids then replicate extrachromosomally (similar to plasmids)
- few copies made per cell (normally one)
19
Q
BACs
A
- 100 to 200 kb
- dna inserted
- plasmid introduced to bacterium
20
Q
3 METHODS OF INTRODUCING RECOMBINANT DNA TO BACETRIA
A
- transformation
- transduction
- infection
21
Q
Transformation
A
- bacteria incubated with recmb dna
- recomb becomes plasmid chromosome
INCUBATION: calcium chloride solution or electroporation
plasmids - solution
bacs - electroporation
22
Q
TRANSDUCTION
A
- recomb dna combines with phage proteins
- produce virus with mostly non-viral DNA
- inject dna into bacterial cells
fosmids
23
Q
infection
A
- produces recomb phage particles
- repeated infection - plaque of A phage particles
- Contains viral genes to make new infective particles
24
Q
Genomic Library
A
- RE break human genome
- each fragment inserted into different copy of vector
- each fragment represented 5 times to ensure all regions included
SHOWS ENTIRE GENOME
25
cDNA library
- mRNA purified from cellular source
- converte dto cDNA
- inserted into vector
-add restriction sites - dna ligase links dna
linkers/adapters to cDNA
- cdnas digested with RE - staggered ends
- requires 10 000 s- 100 000 s independent cDNAS clones
SHOWS ONLY GENES EXPRESSED IN SAMPLE CELLS
26
Identify gene of intrest from dna library
COLONY/PLAQUE HYBRIDIZATION
- similar to southern blotting
- transfer colony from petri dish to membrane
- membrane incubated in single stranded probe solution
(probe usually cloned piece of dna with complementary sequence to target)
- probes identified by autoradiography = target gene identified
27
Genomic clone uses
- Ins 1 contains all regulatory sequences for normal expression in mice
- genomic clone can be expressed in mice
- genomic clone can't be expressed in bacteria - bacterial proteins don't recognize eukaryotic transcriptional sequences and don't splice
- if made with vector with sequences it could be expressed
28
cDNA clone uses
- ins 1 cDNA clone can't be expressed in mice - lacks regulatory sequences for transcription (not transcribed to mRNA)
If it was made via vectors with the sequences it could be expressed
29
PCR cloning
- PCR primers have restriction sites at 5' end
- after digestion with RE, it can be ligated into vector with same restriction sites
- limited to 2kb
- stitch together multiple (order detrmined via restriction sites at ends) to get longer products
30
DNA assembly methods
- construct large genomic regions and cDNAS, whole chromosomes and genomes
1. Gibson Assembly
2. Assemble parts from different genes
3. Fusion protein
31
Gibson Assembly
- piece together 15-40 bp regions of sequence similarity at ends
- incubate fragments with 3 enzymes
1. endonuclease (chew back 5' ends)
2. DNA POL
3. Ligase
- join fragments at any position (not restricted by RE sites)
32
Joining fragments from multiple genes
Common: transcriptional factors + cDNA
- regulatory mouse insulin gene (only expressed in B pancreas cells)
- cDNA for reporter gene (encodes easy to detect protein)
- expressed in mice
- b cells in pancreas identifiable (only genes to express protein)
GFP (green fluroescence protein) encodes small protein that glows green under uv light
33
Fusion Protein
Expresses single protein ( insulin and GFP amino sequences)
- assemble insulin gene with GFP cDNA so protein-coding regions are translationally in-frame
- insulin protein now tagged
- also tag with epitope tags
34
Dideoxy Sequencing (Sanger sequencing)
Small scale sequencing
- uses ddNTPs
- added to growing DNA chain
- block continued DNA synthesis
requires 4 reactions each with...
- dna segment
- radiactive dna primer
- dna polymerase
- 4 dNTPs
- small amount of one of ddNTPs
1. dna polymerase adds dNTPs to 3' end of primer
2. about 1 in 300 times a ddNTP is added instead and synthesis is terminated
added based on base pair therefore location of termination when a pair has been made
(termination in ddGTP tube means it ended at a C nucleotide)
3. gel electrophoresis
4. autoradiagrophy
Bands at bottom = sequence closest to primer
Read in 5' to 3' direction from bottom to top
READING COMPLEMENMTARY STRAND
up to 200 bp
35
Automated sequencing
Uses automated electrophoresis with fluorescently dyed ddNTPs instead
- up to 1000 bp
- single reaction
- unlabelled primer
- each ddNTP labelled with different colour dye
- seperated by size via gel electrophoresis
- laser detects fluorescence
- each peak represents a nucleotide
if there is a single nucleotide polymorphism (reads 2 nucelotides in same location) = that bp different on each allele
36
Transgenes
Transfer of vector into eukaryote
- Chemical
- Biological
- Physical
37
Chemical transgenesis
co-precipitation with mineral
- calcium phosphate
phospholipid vesicles
Cell englufs from environment
38
Physical transgenisis
Electroporation
- electrical field creates tiny holes in plasma membrane
Biolistic particle delivery systems (gene guns)
- bombard cell with dna-coated metal particles
Microinjection
- fine point needle directly injects cell
39
Biological transgensis
Use bacterium or viruses
- transformation
- transduction
- infection
40
Results of transgensis
Gene enters nucleus
- can either replace resident gene via homologous recombination
- OR insert ectopically (insert at other location of genome)
41
Genetic engineering S.cerevisiae (yeast)
- YIps = yeast integrative plasmids = simplest yeast vectors
- onmsert chromosomes via homologos recombination into resident gene
- can be used to delete or substitute a mutant allele
- double or single crossover
double = replacement
single = addition
42
Genetic engineering plants
GMO
- Ti Plasmid
- bacterium derrivative crown gall disease (plant grows tumours)
- due to a 200kb circular dna plasmid (Ti - tumour inducing)
- bacterium infects plant and transfers and inserts part of Ti plasmid
- inserted region = T-DNA
- any dna inserted between Tdna borders can be inserted into plants
- replace dna and add marker
- introduce to plants
infect segments of plant tissue and place on medium with Kanamycin
- plant cells that aquired the kanR gene through transgensis undergo cell division
- they grow into a clump - transfered to soil
43
Genetic engineering in animals
1. Nematode (C.elegans)
- microinjection
- as plasmids, fosmids, typically
- Injected into gonads
- forms multicopy extrachromosomal arrays
- exist outside chromosomes
- some are inherited but with poor efficiency
2. Mouse (Mus musculus)
ECTOPIC INSERTIONS
- inserted randomly in gemone
- solution injected into fertilized egg
- injected eggs inserted into mouse
- positive mice mate and create stable trasngenic lines
PROBLEMS
a) expression pattern can be abnormal due to position effects of local chromatin environment
b) DNA rearrangements can occur inside multicopy arrays
44
Gene targetting
Eliminate or modify gene function
Gene replacement
- mutant allele subsituted by WT
- avoid positin effects and rearrangements
Gene Knockout
- inactivate a gene
- replace it with an inactive versino
45
Process of Gene Knockout in ES cells
neomyacin resitant = neoR = marker
Vector contains hepes virus (tk) gene
WANT NEOR TK-
es cells have neos tk -
1. clone gene into vector tk+ gene
2. Inject into ES cells
3. 3 possible outcomes
a) Homologous gene = chromosome with target insertion (neoR tk-)
b) non homologous = random insertion (neoR tk+)
c) non homologous = unchanged chromosome (neoS tk-)
4. Select cells with gene knockout
- analog of neoS to kill neoS (kills cells that did not inherit vector dna)
- solution to kill tk + (kills cells eing randomly integrated)
- add to medium
- cells with targeted mutation will remain
46
CRISPR
Double Strand Breaks = DSBs
Repaired by
NON HOMOLOGOUS END JOINING (NHEJ)
- can lead to inserted/deleted nucleotides
OR by homologous recombination (HR)
- no errors - uses homologous donor DNA
THEREFORE: direct DSBs, use NHEJ to inactivate gene, use HR to insert DNA
3 technologies to create site-speciifc DSBs
a) zing-finger nucleases (ZFNs)
b) transcription activator-like effector nucleases (TALENs)
c) CRISPR-Cas
a and b are proteins with 2 functional domains
1. dna binding activity to bind a specific dna sequaence
2. non-specific endonuclease activity
CRISPR-cas base pairing tragets cas endonuclease to generat eDSB at specific place - uses non-coding rna
- Cas9 produces DSB in foreign dna that has 20 nucleotide complementary sequence next to NGG protospacer adjacent motif (PAM site)
2 plasmids introduced
- express cas9 protein
- express sgRNA with guide sequence
Introducing a donor plasmid will lead to HR
47
Study of phenotypic consequences of loss of 1/2 insulin genes in mice
Null Ins1 generated by CRISPR DELETION OF INS1 GENE
or
replaced by reporter gene
48
Manipulation of Gene expression via CRISPR
- mutant Cas 9 lacking endonuclease activity
- sgRNA
together:
transport any protein/protein domain to specific place in genome
Bio 205
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