1. cDNA, vectors and libraries
1.1. Complementary DNA
1.1.1. A DNA copy of an RNA molecule. Constitutes the coding region of a gene but does not contain promoter sequences or introns. mRNA cannot be cloned, must be converted to DNA, one strand of RNA complementary to RNA strand created with reverse transcriptase (requires an RNA template, a primer (synthetic oligo-dT complementary to the 3- poly(A) sequence of eukaryotic mRNAs) and dNTPs and buffer)
1.1.2. Single stranded DNA can be converted to double stranded DNA by DNA polymerase, hairpin loop as a primer for second strand synthesis. Hairpin cleaved by treatment with S1 nuclease. Fidelity lower than DNA polymerase as lacks proofreading ability. cDNA can be used to express eukaryotic genes in bacteria (lacks introns and eukaryotic regulatory sequences).
1.1.3. Can be used to generate cell-type collections of cDNAs by using whole population of mRNAs purified from a particular cell type. B/c of different expression patterns, a collection of cDNAs form one cell type may differ from a cDNA collection from a different cell type.
1.2. Vectors
1.2.1. Plasmid
1.2.1.1. Small circular bacterial molecules that can carry foreign fragments for amplification or expression. Have origin of replication (replicate well in E.coli without integrating into genome, high copy number thus amplification of inserted gene. Have selectable marker so that E.coli transformed with plasmid will survive in circumstances that kill non-transformed cells (antibiotic resistance genes/fluorescence markers as addition).
1.2.2. Expression
1.2.2.1. Also contain shine-dalgarno sequence, (ribosome binding site). plus transciptional terminators and translation stop codons, to allow correct transcription and translation of the foreign DNA
1.2.3. Variations on plasmid vectors
1.2.4. Other vectors
1.2.4.1. Bacteriophages
1.2.4.1.1. Viruses that naturally infect bacteria so serve as very efficient vehicles for getting foreign DNA into bacteria. 16x higher efficiency than in plasmid vectors.
1.2.4.2. Cosmids and fosmids
1.2.4.2.1. can take large amounts of insert DNA, but are based on plasmids-widely used in genome projects
1.2.4.3. Bacterial artificial chromosomes
1.2.4.3.1. i. possess bacterial origins ii.selectable markers iii.can contain very large DNA inserts
1.2.4.4. Yeast artificial chromosomes
1.2.4.4.1. linear and have centromeres, telomeres and replication origins as well as an autotrophic selectable marker. can accept Mb of DNA
1.2.5. Made of DNA and are used to "carry" DNA directionally into a specific host cell. Different types according to ease of manipulation and amount of foreign DNA that can be accommodated in vector. Normally have restriction sites suitable for insertion of foreign DNA, a selectable marker, replication origin to ensure their propagation in host cell.
1.3. Libraries
1.3.1. Genomic
1.3.1.1. Made up of restriction enzyme-generated fragments of genomic DNA of an organism, inserted into vector cut with same restriction enzyme. DNA fragments include promoter sequences, introns, and other non-coding DNA, in addition to coding sequences. Genomic libraries are specific for particular organism but do not usually differ between different cell types of that organism.
1.3.2. cDNA
1.3.2.1. Comprise vectors containing cDNA copies of virtually all the mRNA molecules present in an individual cell type. Will vary between cell types, since cDNA is generated from mRNA. Contain only coding regions, and 5' and 3' untranslated sequences, not promoters or introns or intergenic sequences. Many cDNAs in a library will contain an intact 3' end of the coding sequence but will not necessarily be full length as reverse transcriptase has limited processivity and tends to drop off the RNA template before reading the 5- end.
1.3.3. Expression
1.3.3.1. Designed for expression of foreign DNA in, for example, bacterial host cells- hence only cDNA because bacteria cannot process transcripts from eukaryotic genomic DNA due to presence of introns, regulatory sequences and non-coding DNA. cDNAs are cloned downstream from an inducible promoter (eg. from the lac operon) and ribosome binding site, in an expression vector. E.coli are transformed with the library.
2. Gene cloning
2.1. Now that many genome sequences known, new genes are cloned by PCR directly from cDNA or genomic DNA (gDNA). Where genome sequence is unavailable, PCR can still be used with degenerate primers, or traditional library screening is used.
2.1.1. Cloning gene according to protein sequence
2.1.1.1. The protein is purified according to activity, or as a band on a sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE). Partial amino acid sequence is determined. 1. Edman degradation generally used. 2. more sensitive techniques such as mass spectroscopy allow amino acid determination from much smaller amounts of protein. Sequence of the DNA encoding these amino acids deduced, although degeneracy of genetic code means that there are always several possible DNA sequences. DNA sequence is synthesized in vitro as a set of degenerate oligonucleotides that includes all possible variations of genetic code for each amino acid. The oligonucleotide can be used as a primer PCR too for direct cloning, or to generate a larger more specific probe for library screening.
2.1.2. Cloning by PCR
2.1.2.1. Primers are designed according to genome sequence. (if not known, regions of maximal homology are determined by sequence comparison of the protein or DNA in species from which gene has already been cloned-using bioinformatics software.
2.1.2.2. Oligonucleotide primers are synthesized: 1. these are used directly as primers in a PCR reaction with template DNA. 2. If nucleotide sequences vary widely between different species, inosine can be used in the synthetic oligonucleotide as it will hybridize with all four based of DNA. 3. Degenerate primers can be used if genome sequence is unknown but protein sequence is known. 4. Restriction enzyme sites can be included to facilitate subsequent cloning steps.
2.1.2.3. If cDNA/gene too long to be amplified in a single PCR reaction, several adjacent regions can be amplified in individual PCR reactions: 1. PCR products are cleaved with restriction enzyme to produce compatible ends 2. restriction fragments are ligated together to generate full length construct.
2.1.3. Cloning by phenotype
2.1.3.1. A cell line defective in a specific gene function is used, eg. human disease zeroderma pigmentosum. These cells are transfected with a cDNA expression library in shuttle vectors. Correction of defective phenotype is assessed-this is called complementation assay. Plasmid vector DNA is isolated from transfected cells showing the corrected phenotype. Isolated DNA is then amplified by transformation into E.coli and growth in large-scale culture, then purified and sequenced. This process is more frequently used to isolate higher eukaryotic homologues of yeast genes by complementation of the relevant yeast mutant phenotype.
2.1.4. Cloning by library screening
2.1.5. Positional cloning
3. Getting DNA into cells
3.1. Once DNA of interest cloned, it's usually necessary to insert it into living cells for plasmid amplification, recombinant protein expression, or into eukaryotic cells for studying gene activity or protein function.
3.1.1. Biolistics
3.1.2. Transforming E.coli
3.1.2.1. Uptake of foreign DNA into E.coli is called transformation. 1. bacteria are incubated on ice with DNA in the presence of RbCl and CaCl2-these help to neutralize negative charge on DNA and cell membrane, and low temperature reduced membrane fluidity 2. DNA associates as membrane in regions known as adhesion zones. 3. cells are exposed to transient heat shock-this creates a thermal gradient across membrane that encourages DNA uptake. 4. cells must be allowed to recover before antibiotic selection is imposed. 5. Alternatively, electroporation can be used whereby very high voltage applied across membrane for very short period of time, encouraging DNA uptake. Suitable E.coli strains must be used eg proteases.
3.1.3. Getting DNA into mammalian cells
3.1.3.1. Uptake of foreign DNA into mammalian cells is called transfection. Mammalian cells take up liquids by micropinocytosis-they can also take up tiny particles through this route. 1. DNA incubated with calcium phosphate (which precipitates) and this precipitate is then taken up into cells 2. efficiency can be improved by using liposomes or polyamines. Electroporation can be used to stimulate uptake of foreign DNA into mammalian cells.
4. Protein analysis
4.1. Edman degradation
4.2. Measuring protein concentrations
4.3. Protein gels
4.4. Two-dimensional IEF
4.5. Proteomics
5. Immunological techniques
5.1. Antibodies
5.1.1. Proteins produced by the immune system that have high affinity and high specificity for a particular antigen. Majority of antibodies used in biochem techniques are IgG molecules with two heavy and two light chains, linked by disulfide bonds. IgGs have two binding sites per molecule formed by the variable Fv region within the Fab domain. All anitbodies of a particular organism species share a common region (the Fc domain), which can be recognized either by antibodies from a different species, or bound directly to protein A. Fc domain can be swapped using molecular tech. to prevent antibody being recognized as foreign (eg. using human Fc domain on a mouse monoclonal antibody to allow its use as a drug in humans).
5.1.1.1. Monoclonal
5.1.1.1.1. Produced from a single B cell clone and so all recognize the same epitope on the antigen. they are secreted by immortal cells grown in tissue culture and so provide theoretically infinite supply of antibody.
5.1.1.2. Polyclonal
5.1.1.2.1. Recognize same antigen but are produced by different B cell clones. and my recognize different epitopes on the antigen
5.2. Chromatin immunoprecipitation
5.3. Radioimmunoassay
5.4. ELISA (enzyme-linked immunosorbent assay)
5.5. Western blotting
5.5.1. Used to isolate protein of interest (and any associated protein partners) from a complex mix of proteins
5.5.1.1. 1.Antibodies specific to protein of interest are incubated with complex mix of proteins eg cell lysate. 2. antibody molecules are immobilized via their Fc portion (using protein A attached to large sepharose beads). 3. Beads are collected by gentle centrifugation, precipitating also the bound protein 4. after washing proteins can be released from beads by boiling in SDS with reducing agents, and proteins separated on SDS-PAGE (this treatment breaks apart the heavy and light chains of antibody molecules as well as dissociating protein of interest from the antibody). 5. Has been used to identify components of, for example, the Fanconi anaemia complex-antibodies against one component co-immunoprecipitate other components of complex.
6. Genome sequencing
6.1. Shotgun sequencing
6.1.1. Chromosomes are fragmented and inserts cloned into BACs, both ends of each BAC are sequenced directly to give a BAC fingerprint. A "seed BAC" is chosen and the 150 kb insert is fragmented and inserted into plasmid or M13 libraries. Each insert is sequenced and sequence information is assembled using bioinformatics. These are compared with BAC fingerprints. Contiguous BACs with minimal overlap are sequenced (via fragmentation and insertion into plasmid or M13 libraries)-this is known as BAC walking
7. DNA sequencing
7.1. Dideoxy chain termination sequencing (Sanger sequencing)
7.1.1. 1.DNA to be sequenced is purified (denatured if double stranded first). 2.This DNA used as a template in a DNA replication reaction using dNTPs spiked with low concentration of dideoxyribonucleotide triphosphates (ddNTPs) as random chain terminators (lack 3' -OH on ribose.
7.1.2. Reactions contain: 1.purified DNA template 2. synthetic single-stranded short oligonucleotide primer complementary to known region of DNA 3. mixes of all four deoxynucleotide triphosphates 4. low concentration of dideoxy form of all nucleotides, one per reaction in radiolabelled systems/all 4 together in modern fluorescence base systems. 5.modified DNA polymerase that will incorporate ddNTPs as well as normal dNTPs. 6.radiolabelled dATP in traditional radiolabelled sequencing reactions OR ddNTPs each labelled with a different coloured fluorophore in modern fluorescence reations
7.1.3. Process: New complementary strand polymerized by DNA polymerase, synthesis stops when dideoxy nucloetide has been incorporated, this produces a range of different sized fragments. Fragments separated by size on gels, DNA bands detected on X-ray film/lazer excitation, sequence then read according to position DNA fragments migrate
7.2. Pyrosequencing (454 sequencing)
7.2.1. DNA sequenced in real time by directly analysing incorporation of new dNTPs during DNA synthesis, DNA template with primer annealed is immobilized in a nano flow cell with a light detector, reaction mix includes DNA polymerase, ATP sulphurase, luciferase, buffer and Mg2+, Each dNTP in turn flowed over the immobilized template with washing in between
7.2.2. When nucleotide incorporated into DNA: 1. pyrophosphate is released, 2.PPi is converted to ATP by ATP-sulphurylase 3.luciferase used the ATP to generate light 4.amount of light given off proportional to the number of nucleotides incorporated when one type of dNTP is flowed through the cell, repeated for each dNTP (very rapid reaction).
7.3. PCR
7.4. DNase 1 footprinting analysis
7.5. Site directed mutagenesis
8. DNA manipulation and analysis
8.1. DNA concentration determination
8.2. Restriction enzymes
8.3. DNA ligase
8.4. DNA gels
8.5. Southern blots
8.5.1. A method for determining the presence and copy number of a DNA molecule of which at least some of the sequence is known
8.5.1.1. 1. DNA is fragmented using restriction enzymes and separated on an agarose gel-if fragments are very large gel may be exposed to acid to break DNA within individual gel bands to smaller pieces to increase efficiency of transfer. 2. DNA is transferred to nitrocellulose/nylon membrane by capillary action in alkali buffer to denature DNA, or in neutral buffer. 3. Membrane is baked or UV crosslinked to fix the DNA onto the membrane. SIngle-stranded radiolabelled DNA probe complementary to DNA on blot is incubated with membrane. 4. DNA can be radioactively labelled on either the base (3H-thymidine) or the phosphate. 5. Unbound probe is washed off. 6. Blot is exposed to X-ray film and a band is observed where the probe has bound to the blot.
9. RNA manipulation and analysis
9.1. RNA isolation
9.2. RNA concentration determination
9.3. Northern blotting
9.3.1. A method for determining expression levels and transcript size of an RNA product of a gene of which some of the sequence is known.
9.3.1.1. 1. Total RNA extracted from cells and separated on agarose gel in presence of formaldehyde. 2. RNA transferred from gel to nitrocellulose/nylon membrane by capillary action in neutral buffer. 3. membrane baked or UV crosslinked to fix RNA onto membrane. 4. single-stranded DNA probe complementary to RNA of interest incubated with membrane-probe is radiolabelled or fluorescently tagged 5. unbound probe washed off and blot exposed to X-ray film-a band is observed where the probe has bound to the plot 6. norther blot can be used quantitatively to measure the level of gene expression. Size of transcript can be determined by position band has migrated on gel compared with markers-this allows determination of transcript size and any alternative splice variants.
9.4. RT-PCR
9.5. Microarrays
9.5.1. These provide a powerful method for comparing relative levels of expression of >10,000 genes at a time on a single glass slide
9.5.1.1. cDNAs or oligonucleotides are printed onto a glass side using a modified inkjet printer that uses DNA rather than ink. To test relative gene expression, cDNA is prepared from mRNA extracted from different types of cells, or the same cells exposed to different conditions (drug, cancer vs normal, old vs young etc.). Test (T) cell cDNA compared with reference (R) cell cDNA. T and R cDNAs are labelled with different fluorescent tags (red for test, green for reference). T and T cDNAs are hybridized to chip: if gene expressed, cDNA binds and fluoresces.
9.5.1.1.1. i. T cDNA binding: red signal (gene expressed only on treatment)
9.5.1.1.2. ii. R cDNA binding: green signal (gene expressed only on control)
9.5.1.1.3. iii. T and R both bind: yellow (gene expressed under both conditions).
9.5.1.1.4. Can measure both up and down regulation of gene expression in cell of interest vs. reference cell.