Cloning and Gene Manipulation

Restriction enzymes - proteins that cut dsDNA

In bacteria - used as defence mechanism against foreign DNA from viruses - chops it up

Sequence specific endonucleases cut in different ways:

• Blunt ends
• 5' overhangs
• 3' overhangs

Host bacterial DNA protected by methylation

To cut DNA - 2 incisions - once through each backbone

Palindromic recognition sites often

Some endonucleases cleave certain number of bases away from recongition sites 

= convienitent generation of define DNA fragments with known ends

• Completely digest DNA w/ restiction enzymes
• Load digest into wells of agrose gel
• Gel - voltage applied
• DNA -ve charged - move from cathode to anode
• Smaller pieces move faster = seperate on site
• Gel stained with fluorescent dye - binds DNA 
• Observed on UV light box in comparison with size marker
• Bands of interest cut out and isolated

If DNA restriction sites known = compatible restriction fragments from different DNA sources

Use compatible sticky ends - anneal using hydrogen bonds - not stable (weak H bonds)

Ends joined covalently using DNA ligase

Two different restriction enzymes used - add directionality to DNA = read and code

Used to visualise specific DNA fragments

Polynucleotide kinase - enzyme adds 32P to 5' end of DNA

Labelling important for DNA footprinting

• Heat DNA - break hydrogen bonds
• Probe DNA with primers
• Primers anneal to single stranded DNA - DNA Pol added = DNA synthesis
• Visualise DNA by modifying bases with radioactive P, biotin or digoxigenin

Digoxigenin (DIG) - modified dTTP thats highly antigenic - high affinity to specific anti-DIG antibodies = detected in labelling

Used to determine where genes are on chromosome during metaphase (chromosomes pulled apart)

Different probes w/ fluorescent antibodies (different colours) used under stringent conditions to visualise genes

Genes can be identified and localised

Stringent conditions:

• Hybridisation in 50% formamide at 42C
• = Formamide stabilised single-standed DNA
• Only completely complimentary DNA stands base pair

Non-stingent conditions

• Hybridisation in 50% formamide at 35C
• Causes imperfect base pairing
• Base pairing between similar DNA strands

Used to transfer DNA to nitrocellulose paper - no diffusing away (FIXED)

• DNA suctioned onto paper as water moves by osmosis, drags DNA with it
• Alkali solution - denatures DNA = probing occurs
• Paper shaken in sealed bag with labelled probes
• Labelled probes hybridized to complementary DNA bands = visualisation by autoradiography
• Identify specific DNA fragments by choosing specific probes to bind

Using non-stringent conditions with southern blotting = help format DNA family trees 

Northern blots - detect RNA

Southern blots - detect DNA

Western blots - detect proteins

Transcription factors - proteins that can bind to DNA

DNA footprinting determines the site that transcription factors bind

• dsDNA fragment prepared - end label one strand with 32P
• Transcription factors allowed to bind to DNA
• Cleave DNA at random (w/ DNase) - except at area where protein is
• Proteins can be removed and DNA denatured and seperated
• X-ray film of it shows ladder - one area with no bands = DNA protected by transcription factor = FOOTPRINT

TF not all same size and shape - need to determine which TF has bound to which DNA section

• Radioactively labelled DNA fragments w/ TF binding sites
• DNA incubated w/ extract from tissues/cells of interest
• Samples run on polyacrylamide gel - more robust for TFs bound
• Pieces of DNA w/ larger/higher numbers of TFs bound move more slowly
• Gel exposed to x-ray film

Vector - DNA molecule used as vehicle to artifically carry foreign DNA into cells

Plasmid - small, circular dsDNA in bacteria

Plasmids used by bacteria to transmit beneficial genes e.g. antibiotic resistance

Artificial plasmids can be vectors in genetic engineering - inserts of up to several kilobases in size

• Plasmid cloning vector
• Cleavage with restinction endonuclease
• DNA fragment linked in with DNA ligase = recombinant DNA
• ds recombinant plamid introduced into bacterial cell (TRANSFORMATION)
• Bacterial cell cultures and produces millions of bacteria
• Copies can be purified and isolated from lysed bacterial cell

Genomic DNA - high molecular mass if good quality 

Human genome difficult to cope with in research - genomic library used

Genomic library - collection of bacterial/phage clones. Each clone carries copies of particular DNA segment from foreign genome in plasmid. 

Complete genomic libraries - foreign DNA segments cover entire genome

• DNA digested so average fragment size suits vector used
• Fragments ligated together = bacterial vector called Bacterial artificial chromosomes (BAC)
• Each bacterium takes up recombinant BAC w/ different insert = 1 primary clone in library

Libraries stored in multiwell plastic plates - each clone occupies one well

To find a gene of interest in genomic library - complementary labelled probe used (shaken in bag w/ special treated memebrane - break DNA)

Non-stringent conditions to find related genes to gene of interest

In addition to exons, libraries will contain introns and intergenic regions

If only interested in exons - library of mRNA used:

• Complementary DNA (cDNA) synthesised from mRNA isolated from tissue cells
• Reverse transcriptase (RT) synthesises cDNA
• RTs - viral enzymes that make DNA from mRNA using stretch of dTs as primer
• mRNA degraded by RNAse
• DNA polymerase synthesises second strange using primer = cDNA
• cDNA = coding sequence w/ no introns = cDNA library

PCR used to copy specific section that doesn't contain convenient restriction sites

Chain reaction similar to nuclear chain reaction but DNA olignucleotide primers added

DNA polymerase requires them

Copying stopped at certain time - only desired region is copied/amplified

Fragments of similar size cannot be distinguished using electrophoresis

DNA sequence confirms fragment identities and obtains structural information of genes

Dideozyribonucleosides TP (ddTP) - chain terminator - prevents extension at 3' end of DNA

Adding small amount of DNA blocks futher growth

4 different ddTPs can be added - stop at individual points

Seperate strands on size will form ladder = read to form DNA sequence

Original method - radiolabelled nucleotides - formed 1 ddTP per n per track on gel

Fluorescent probes used commonly + sample run on single capillary

Same as Southern blotting - RNA

RNA seperated by size w/ gel electrophoresis and blotted onto membrane

Specific pieces detected with labelled probe

Can be used to determine RNA size/quantity 

See how much RNA expressed relative to control w/ addition of something to transcription factor e.g. siRNAs that stop transcription

Actin often control in experiments

• mRNA prepared from tissue of interest
• cDNA synthesised from mRNA w/ reverse transcriptase
• PCR used to amplify cDNA using gene-specific primers
• cDNA run on gel - compare level of expression between tissues
• Thicker bands = more cDNA in them

Can also be done quantitatively - see how levels change over time or between tissues = qRT-PCR

Antisense RNA - single-standed RNA complementary to strand of mRNA

Introducted to a cell to inhibit translation bu physically obstructing translational machinery

Effect related to siRNA - best method at looking directly at mRNA levels in tissues

To make asRNA probe:

• Gene of interest cloned between 2 bacteriophage promoters 
• 1 promoter - use polymerase to make synthetic mRNA w/ polyA tail
• Other promoter - make antisense probe - labelled and hybridised to required mRNA

Example - Drosophilla Embryo Development

• Make antisense RNA probe to Delta C mRNA
• Probe base pairs to target mRNA in Drosophila embyro
• Wash away excess probe
• Detect probe by antibody - activated by an enzyme = purple
• Enables visualisation of where gene expressed most and at what time in development

Example - Early Zebrafish embryo

• Antisense RNA probes against 5 different mRNAs of different colours
• Shows levels expressed of different genes in different embryo areas

Examples show importance of spatial expression of genes to build organism - also linked to developmental stages

Involves proteins - can be used to see if proteins are where mRNA is

Seperate proteins based on size using SDS-PAGE method (verticle - proteins forced by gravity)

Proteins 3D and different charges:

• Heating - break H bonds
• Mercaptoethanol - breaks covalent bonds
• SDS - gives each molecule a negative charge = balances charges

Proteins seperated by molecular weight and transferred to membrane to bind in place

Specfic proteins detected w/ primary antibodies and labelled by secondary antibody

Markers include fluorescent molecule or enzyme

• Gene of interest identified
• cDNA to gene created
• Protein that is coded for by gene expressed in cells
• Recombinant protein-of-interest created
• Immunised rabbit generates antibodies - complementary to protein
• Antibodies have proteins attatched = identified

Used to visualise how organism/tissues react when genes are switched on/off or removed/added

Inserting lacZ or gfp (green) genes into intron of gene = becomes fused coding region and exon

Gene turned on = increase in colour/fluorescence

Identifies when and where genes expressed

Reporter can do strange things to organism = mutant genes used instead w/ assumption that gene change not lethal

Example - Noggin in frogs

• Noggin mRNA injected into frog egg at site different to normal expression
• Noggin detected w/ antisense probe labelled w/ DIG
• Ectopic noggin induces extra body axis - changes polarity

Occurs in oligomeric state proteins

The dominant-negative gene product completes w/ wild type and supresses function of wild-type gene product

Mutation in one subunit = mutation in all protein subunits

DNA profile - small set of DNA variations likely to be different in unrelated individuals = unique to individuals

DNA is different enough to distinguish individuals, unless monozygotic (identical) twins

DNA profiling - uses repetitive (repeat) sequences that are highly variable = variable number tandem repeats (VNTRs)

Particularly short tandem repeats (STRs) - microsatellites/minisatellites

VNTRs that have repeat length 10-100bp

Repeat number - 2 to 100

30 minisatellite alleles for each locus

Minisatellites analysed by Southern blotting using repeats as probes and separating based on size

Different patterns used to identify different individuals

STRs - repeat length 2-9bp

Repeat number 7-40

Hundereds of STR loci in genome

DNA profiling PCR - amplify several STR loci w/ labelled primers - seperated by gel electrophoresis

Advantages - very little DNA required

Taqman probe:

• Fluorescent reporter dye and quencher attatched to probe
• When probe is intact - repoter dye quenched
• During extension, DNA polymerase cleaves reporter dye from probe
• When seperated from quencher (and probe), reporter dye emits fluorescence

Sybr Green probe:

• Denatured DNA - SYBR green released and fluorescence reduced
• In extension, primers anneal and PCR product generated
• When polymerisation complete, green probe binds and increases fluorescence 

Positive reaction detected by accumulation of fluorescent signal

Ct (cycle threshold) - number of cycles required for flourescent signal 

Ct levels inversely proportional to amount of target cDNA in sample

Comparison carried out in comparison to housekeeping gene e.g actin, tubulin