Genetic Methods and Technologies

Question 1

Traditional Cloning

Answer 1

1. Break DNA (vortex, rigorous pipetting, sound waves; uncontrolled). Or use restriction enzymes.
2. Cut vector and gene of interest, to form sticky ends (vector should be around 10 kb or less)
3. If vector is digested with single enzyme, de-phosphorylate to prevent self ligation using CIP (5’-p)
   additional treatments include:
   i) blunting
   ii) A-tailing
   iii) phosphorylation
4. Use ligase to covalently join vector and insert
5. Transform plasmid into chassis of choice
6. select and screen for gene/fragment of interest

Question 2

DNA extraction

Answer 2

Manual:

• Lyse cells (centrifuge, shake, blend etc.)
• Homogenise (with SDS/protein K)
• Add phenol/chloroform (phenol acts on hydrophobic part of protein, forming micelles, denatures protein)
• Nucleic acids are now in soluble phase
• Precipitate DNA with ethanol, purify from soluble phase

Disadvantages: time consuming (multiple centrifugation steps, slow acting phenol etc.)

Silica based DNA/RNA purification kit

1. homogenise/lyse DNA as above
2. Bind to silica column under mild centrifugation
3. wash with buffer a few times to get rid of protein etc.
4. Elute DNA from column with water

Takes around 4 min. Very efficient

Question 3

Molar Ration Insert:Vector

Answer 3

Number of molecules of insert and vector (not just mass/volume)

Concatamers: multiple inserts in a single vector. Happens when Molar ratio of vector and insert are off

Question 4

Double Enzyme Digest

Answer 4

Fixes Orientation
Prevents self-ligation
Uses NEBcutter to identify compatible enzymes
(must be compatible with buffer environment, fit on the plasmid etc)

Question 5

Site Modification

Answer 5

De-phosphorylation of 5’. Vector cannot self-;ligate with 3’-OH. Requires 5’-phosphate from insert. Done using phosphatase.

Blunting of sticky ends:
- Remove of Overhang (Klenow fragment 3’ end, Bean Mung 5’ end)
- Fill in overhang (3’ end not possible, Klenow fragment 5’ end)
The advantage of this is that requires little preparation (can be used in combination with sticky end for directionality)

Blunt-end ligation is usually less effective than sticky-end. (use higher concentration of DNA fragments and ligase)

*Klenow fragment is a part of DNA polymerase 1 that has lost 5’ to 3’ exonuclease (cannot remove primer)

Question 6

NEBcutter

Answer 6

Analyses restriction sites for particular enzymes
Can find compatible restriction enzymes for double digest (temp., buffer, efficiency, *star ability etc.)

*star ability refers to multiple cutting sites.

Question 7

Gibson Cloning

• process
• pros/cons

Answer 7

1. Self-defined 20-40 bp overlap of fragments (in insert and plasmid, or with multiple fragments)
2. chew back at 50˚C with TS exonuclease
3. Anneal at 50˚C with Taq ligase and Phusion polymerase
4. Repair at 50˚C with Taq ligase and phusion polymerase
5. Transform into bacteria, grow, select and screen

Pros:
efficient at creating large DNA from multiple fragments. Multiple fragments at once. Up to 100 kb.
Single reaction

Cons:
becomes unstable wit more than 5 fragments at once. Chewing back can cause nucleotide deletions followed by insertions, leading to frameshift mutations/del mutations etc.

Question 8

PCR Amplification

Answer 8

Primer design
(CG-AT ratio, 18-24 nucleotides, no wrong binding sites etc.)

1. Denaturation (80˚)
2. Annealing (55-60˚)
3. Extension (72˚)

Unsuccessful PCR:
DNA contamination 
Wrong temperature will lead to smearing; too low will cause annealing at the wrong time, too high will cause imprecise annealing. 
[MgCl2] affects annealing: 
Too little= reaction will not proceed
Too much = non specific binding

Question 9

Next Generation Sequencing

Answer 9

Based on multiple shorter reads (100 bp)
Overlap to get sequence
Placed into high coverage contigs

Illumina:
Clonal bridge amplification used to get clusters of fragment that is then sequenced
Employs modified deDNA: 4 dyes with reversible terminators
Extends each fragment from flow cell with illuminating sequence
Each product is removed with denaturation (high temp) to allow for a new read

Question 10

RNA in situ hybridisation

Answer 10

Allows for the localisation of RNA in tissue
(only localisation, no quantitative data)
- binds to target mRNA using DIG-labelled probe
(DIG = tag)
- The excess probe is washed off using ethanol
- Antibody/anti-DIG-Alk-phosphatase
Has substrate that turns blue

Can also use fluorescent tag (Fluorescence in Situ).
The secondary antibody is an amplifier of the signal

Question 11

Southern Blotting

Answer 11

Southern Blot:
- Probe sequence hybridises at specific genomic sequences
- people will have different number of sites that the probe will bind to due to polymorphisms
- can be used to identify different individuals; varying size/number of labels
inexpensive, time consuming, allows for large segment analysis

Labelling probe added
Random Prime Labelling:
- add hexamers to denatures fragment that will anneal at random sites
- extend using radioactive nucleotides
- Denature and repeat
- Radioactive probe is achieved, which can be bound to southern blot and visualised

Question 12

Labelling Probes

Answer 12

Random prime labelling:

• random hexamers anneal, extend/polymerise with radioactive nucleotides
• Repeat
• Radioactive copy of DNA fragment which can now be used as probe

Nick Translation:

• Make nick in DNA fragment using DNAse1
• Use DNA polymerase 1 to recognise nick. It will remove adjacent nucleotides
• It then repairs the entire section with radioactive nucleotides

Use radioactive probes to target restriction sites, and thereby identify recombinant DNA

Question 13

Northern Blot

Answer 13

RNA expression levels detection across tissue
2 bands that are always present: Ribosomal subunits
(these can be used as positive control)

Allows for the identification of alternative splice forms
- using probe that hybridises across exons

Question 14

RT-PCR (q-PCR)

Answer 14

RNA extraction (genomic DNA is contaminant)

• cDNA-synthesis of RNA
• make primers across exons to avoid genomic DNA (does not work on single exon genes + can miss expression of different splice forms)
• RT (reverse transcriptase) anneals to poly-A tail (oligo dnt). Extends to create cDNA/mRNA hybrid

Amplify using PCR

Quantitative measure:
There is a relationship between number of cycles and expression levels in a certain time frame. After that, the relationship is lost.

Using Cybr-green fluorescence tag
- each cycle the flouresence gets amplified
- after a number of cycles, the threshold for seeing the signal is crossed
- the fewer cycles it takes to cross threshold, the higher the expression levels were to start
- can use multiple colours to compare
There is a RT control with genomic DNA to compare

Can also use Taqman
uses 2 tags on primer: fluorescence and quencher
quencher drowns signal until it is cleaved off during extension

Question 15

RNA-seq

Answer 15

All RNA present is sequenced
- used to identify new splice forms
used for microRNA and mRNA –> converted to cDNA first

It has replaced microarrays as it is cheaper and new splice forms /novel genes will show up

Question 16

Immunostaining/

Immunohistochemistry

Answer 16

Immunostaining
1. Make cell permeable to antibodies (i.e. detergent)
2. Use antibody to binds to target
3. Use secondary antibody to amplify signal with tag
(always use separate species to prevent false positives)
Comassie Blue can be used to stain control proteins
Used to see which tissues a protein is being expressed

Immunohistochemistry
Can use multiple tags to identify various cell types within a tissue that are expressing different proteins
Look at co-localisation of proteins.
Tags include green fluorescence and Horseradish peroxide (DAB as substrate, blue stain)
(does not reveal interactions, only localisation)

Question 17

Protein Interactions

Answer 17

Yeast 2 Hybridisate

Has 2 domains Transcription factor (one at promoter region, one that binds to reporter gene)
Make fusion-proteins of candidate proteins (suspected interaction)
If there is interaction, the first that binds to promoter region will bind and cause reporter gene to be expressed
Reporter gene is usually an amino acids
The hybrids are selected for using different growth mediums lacking those amino acids
- selects using autotroph property

Can be used to check protein as well as domain interactions depending on the fusion proteins you use

Question 18

Pull Down Assay

Answer 18

Used to check protein interactions in vitro
- make fusion protein with GST (can be GSH, polyA tail etc.)
Run through column to see which proteins will bind to candidate gene (GSH in this case)
Collect flow through of any bound proteins with wash.
Collect proteins and use western blotting to visualise.

This method is used to detect any protein interactions that may occur in vivo. Useful for detecting novel interactions as you can run cell lysate through it.

Question 19

IP and Co-IP

Answer 19

IP

Uses antibodies to precipitate proteins of interest of cell lysate solution
Uses protein-A to drag down antibody and protein of interest (makes it insoluble)
Centrifuge and isolate precipitate

This is used to detect lowly expressed proteins (which need to be purified to be detected)

Co-IP

Looks at proteins bound to candidate protein that has been precipitated
reveals in vivo protein interactions

Question 20

GAL4-UAS System

Answer 20

GAL4 = Transcription Factor 
UAS = Upstream activation sequence (promoter) 

Two transgenic lines of drosophila

1: GAL4 + native Promoter (upstream of GAL4)
2: UAS + reporter gene

Cross: GAL4 gene and UAS gene

reporter gene is only expressed in cells where the native promoter is normally expressed

Promoter –> GAL4 –> UAS promoter sequence –> Reporter gene

Native promoter causes expression of GAL4, which binds to UAS driving expression of reporter gene.

Shows which cells native promoter is active in
- used to locate cells where target proteins are expressed

Question 21

Post-Translational modifications

Answer 21

modulate protein function 
Intracellular
- phosphorylation 
- acetylation 
- methylation 
- ubiquitinylation 
- lipidation/prenylation 
- ADP-ribosylation 

Extracellular
- glycosylation

Question 22

RNA Scope

Answer 22

Used to detect mRNA expression in cell.
Add preamplifier with specific sequence to tag mRNA. Then use amplifiers with label probes to amplify signal. Can visualise multiple mRNAs in cell using fluorescent microscope

Question 23

Southern Blot

Answer 23

Diagnosis of disease caused by replication events (Huntington, Alzheimer’s etc.)
Check recombination (i.e. in knock out mice)
Detect genomic rearrangements
Forensics

2 individuals will have different restriction sites due to polymorphisms, and will therefore show different bands on gel
After transfer to Southern Blot from gel:
1. Form probe sequence
2. hybridise
3. Allows for detection of specific sequence in genome

Inexpensive. Very time consuming. Large segments of DNA can be analysed (unlike PCR)

Question 24

qPCR/RT-PCR

Answer 24

PCR works on double stranded DNA
- you need to turn RNA into cDNA to detect expression levels (viral load, GMO, SNP genotyping, allelic discrimination)
- To avoid genomic DNA, primers across exons are used
- therefore only spliced cDNA (from RNA) is amplified
- This does not work on single exon gene.
SYBR green is used to detect fluorescence when above threshold level -> Ct is the cycle number this happens

Alternatively Taqman can be used
- fluorescent tag quenched on primer (primer lacks 3’ OH and is therefore not extended initially). Then as DNA polymerase extends from other primers and hits the tag-primer sequence, the quencher and tag is released, increasing fluorescence. The cycle at which this is detected is Ct.

Quantification
- Standard curve method
compare cycle at which threshold is reached to calibrated curve to quantify absolute number of cDNA molecules at start

• Comparative Threshold method
  Use sample of interest and compare to control (such as in drug tests, a treated and non-treated sample)
  Then compare these to a house keeping gene to see ∆Ct between the sample and the house keeping gene.

Question 25

SYBR Green versus Tagman

Answer 25

SYBR Green

• free in solution and then becomes intercalated
• high affinity for dsDNA/
• upon binding, fluorescence increases.
• Cheaper

Taqman

• Sequence specific probe (18-22 nts)
• in free solution, tag is quenched (quencher absorbs most of the fluorescence)
• when Taq polymerase extends from other primers, the quencher and tag are separated
• increases fluorescence
• More expensive. Can be used to tag different RNA products with different colours

Question 26

RNAseq

Answer 26

Converted mRNA, miRNA to cDNA
It is then amplified
Used to detect novel splice forms as all the expressed RNA is amplified (unlike microarrays)

Question 27

Miniprep

Answer 27

The purification of Plasmid DNA from cells/
Preparation for analyses (PCR, Gel electrophoresis etc.)

1. spin down cells, remove growth medium from top layer and re-suspend in buffer containing RNAse (breaks down bacterial RNA)
2. Lyse with detergent (SDS denature protein and solubilises cell membrane; alkaline denature DNA and breaks cell wall). Vortex to homogenise.
   Neutralise with mild acid to precipitate proteins and genomic DNA.
3. Spin and remove precipitate (save supernatant). Collect plasmid using a micro column. Wash with ethanol or high salt conc. buffer.
4. Elute with water/aqueous weak buffer.

Question 28

Blue White Screening

Answer 28

Vector plasmid: Bluescript plasmid (has ampicillin resistance and LacZ gene).
LacZ gene encodes ß-galactose which turns Xgal substrate blue.

The strain of the bacteria lacks functional LacZ gene in its lac operon (LacZ∆MI5 deletion mutation).

Insert: Nrg gene (in the methods lab example). The restriction sites ensure the GOI is inserted in the lacZ gene (disrupts function).

Agar medium: Contains IPTG (protein involved in functional lac operon), Xgal, Ampicillin.

Cells transformed with vector but no insert (ligated vector): survive, blue
Cells unsuccessful transformation: will not grown
Cells with successful vector and insert: growth, white.

Various controls can be done (checks double cut, ligation, transformation, agar culture, ampicillin quality etc.)

Question 29

C. elegant sequencing case study

John Sulston

Answer 29

1. DNA extraction
2. Cloning into vector
3. Cosmid and phosmid (contain large fragments)
4. Addition of random fragments of linearised DNA, ligation of random fragements into separate vectors
5. Uptake into bacteria (very inefficient). Each bacteria can produce many copies of single fragment, a colony can produce 10^12 of each insert.
6. Insert into liquid culture where it can remain for decades.
7. plasmids are used to sub-clone cosmos
8. clonal colonies are organised using the “Golden Path method”. Radio-labelled insert of one will hybridise to adjacent pieces, and then that fragment will hybridise to its adjacent piece (with sufficient overlap). Visualised on matrix (chromosomal walking)
9. YAC contains larger fragments, and therefore is used to span the smaller cosmic contigs for further alignment.
10. The sequencing was performed using automated capillary sequencing.

Question 30

Multi-cellular transgenesis

Answer 30

1) transient transgenesis of a tissue (i.e. CF treatment with vectors)
2) embryonic transgenesis
3) ES cell transfer to enucleated zygote
4) plant somatic cell regeneration

Question 31

Homologous Recombination of insert

Answer 31

Target insert to specific site using homology arms on either site of transgene/positive selection marker. Tk gene placed outside homology arms (selects against non-homologous recombinants by initiating cell death). A method of specific knock out

Question 32

QTL mapping

Answer 32

Quantitative Trait Loci

1. Linkage Map
   - If recombination of trait and other trait/marker gene is 50%, then likely linked on the chromosome.
   - Select parental strains showing clear phenotypic variation
   - Sequence genomes to compare
   - Create many recombinant progenies to locate genotype with phenotype (the gene causing the trait will be close to the linked marker)
   - Segregation markers are used as linkage placed every 10-20 CM (SNP, restriction sites, tandem repeats etc.)
   - If some markers are restriction sites, then that is a way to distinguish between parents and progeny on a western blot
   - SNP detection is done using probe with fluorescent marker and quencher
2. QTL analysis
   - Look for significant association between marker and phenotype in progeny

QTL map is made using MapManagerQTX

3. Identify region associated with trait

• Look at the LOD scores (logarithm of the odds) to determine how likely the gene region is to be the cause of the phenotype
  >3 evidence for linkage
  3>x<2 : inconclusive. more data needed.
  <2 : no linkage
• LOD score is used to narrow region of interest

Question 33

RNA

Answer 33

short hairpin miRNA: precursor of endogenous miRNA formed during transcription of DNA. Temporary. A good experimental tool.

miRNA: short double stranded RNA

short hairpin siRNA: precursor for siRNA
siRNA: short double stranded mRNA (same as miRNA) expect that it is an exogenous experimental tool. It is eventually degraded.

Question 34

Transposon

Answer 34

31 bp inverted repeats. These are recognised by transposase enzyme. To use transposons for transgenesis, you need the 31 bp set, and the transposase gene. Placed on 2 plasmids and inserted into embryo germline. Marker gene in drosophila (white eye functional gene = red eyes). Since the plasmids are in the germline, and you mate P generation with homozygous white, F1 will have white eyes. You then cross these with white eyed flies as well. Pick out the red eyed flies of F2 as they will have the gene of interest.

Question 35

Plant Transformation

Answer 35

Agrobacterium Tumafaciens (Ti plasmid). Contains viral TDNA which also encodes oncogenes - these are removed from plasmid to not harm the plant. You use virulence genes (will insert into plant genome) and transgene/marker gene on two plasmids (as Ti plasmid is too large to work with). Transform into agrobacterium.

Take plant sample and culture overnight with agrobacterium. Select successfully transformed cells (kanamycin, bar etc.)
Grow into new plant using hormones (auxin and cytokinin)
Remove leaf and use PCR to check for transgene.

Floral dip method:
Soak flowering buds in transformed agrobacterium and detergent (detergent makes cells more receptive). Let flower and seed. Plant seeds in medium that allows for selection (herbicide in soil etc.)

Plant Selectable markers

• npt II (neomycin phosphotranstace which confers resistance to kanamycin antibiotic)
• ngpt
• bar gene (resistance to herbicides)

Question 36

Cre-lox system

Answer 36

Tissue specific knock-out. A method to study lethal mutations (knock-outs).

1. Cre is a bacteriophage enzyme. It cuts DNA at specific sites indicated by LoxP sites, and ligases ends.
2. Tissue has target gene with gene of interest planked by LoxP sites. Organism must be transgenic for Cre.

Orientation of LoxP sites

• same orientation results in deletion
• opposite/towards each other results in flipping the Gene + LoxP sites.

Can be used to target specific exons using Cre enzyme. Transgenic mice (ex.) will only be affected in tissue where Cre is expressed. This can be controlled by inserting Cre gene behind promoter of gene only found in target tissue. (neuronal, heart etc.)

Question 37

Y2H (limits, uses, approach)

Answer 37

Original approach:
Based on Gal4 possessing two domains (bind to UAS promoter and to DNA polymerase I), that perform their function even when not located on same protein. Fusion proteins of each domain, that interact (if the 2 fusions interact) cause the domains to be present together, driving the transcription of reporter gene.

Limitations:

• proteins need to be able to enter nucleus
• bias towards high affinity interactions
• false negatives (of weak interactions unnoticed)
• In library method, false positives can be reduced by coupling to 2 reporter genes. Need to be not just random interaction to drive 2 expression systems.
• Self-activation causes false positive
• False positives can occur if there is high conc. of bait-prey, found in not natural compartment of cells/cell types
• Proteins that require extensive post-translational modification is hard to work with fusion model

Other model systems have been put in place where you detect functions upon interaction of the 2 fusion proteins (membrane recruitment, protein dimerisation, inhibition etc.)

Question 38

Primer Design

Answer 38

• C/G clamp at 3’ end
• C/G:AT ratio of 40-60 percent
• Tm of forward and reverse within 5˚ of each other (and between 50-60)
• 18-24 nucleotides long
• Needs to have restriction recognition sequence in
• Add “stuffer region” before the restriction recognition sequence
• No more than 3 complementarity between primers, and with itself

Question 39

Transformation efficiency

Answer 39

number of cells/colonies with successful uptake/Amount of DNA used for those cells

Question 40

Nanodrop

Answer 40

A260 = dna 
A280 = protein 

amount of DNA: A260 x 50 ng/ul x dilution factor

A260/A280 = 1.8
*should be around 1.8-2.0. Less than 1.8 indicate impure

Question 41

Vector

Answer 41

Ideally less than 10 kb, as larger is difficult to work with and can be unstable.

Some plasmids encode own enzymes for replication, others insert themselves into chromosomes, and some use host enzymes while remaining in plasmid form.

Integrative plasmids = episomes

Yeast has naturally occurring plasmid 2 um circle

Lambda and M13 are bacteriophages that can act as cloning vectors

Question 42

DNA extraction from culture

Answer 42

Grow bacterial cells in broth until dense (measured by optical density)

Centrifuge broth tube to collect pellet of bacterial cells at bottom.

Resuspend in solution with EDTA + SDS and/or lysosomes to lyse cells - EDTA removed Mg2+ needed to maintain bacterial envelope and inhibits cellular enzymes that might degrade DNA. SDS disrupts lipids of membrane.

De-proteinize lysate using phenol/chloroform, which precipitate protein. Can also add protease here to limit repeating this step. mRNA is sometimes removed in this step too. Use Ribonuclease to remove all RNA

Nucleic acid material will be found in aqueous phase

Question 43

DNA purification

Answer 43

Removal of proteins phenol, chloroform and proteases.
Removal of RNA using ribonuclease.

Ion-exhange chromatography to purify further. Negatively charged DNA/RNA will bind to resin column. Salt breaks this interaction - the higher salt concentration, the more tightly bound (DNA) will be eluted.

Ethanol precipitation is another way of purifying as DNA can be centrifuged out of solution.