recombinant DNA technology Flashcards
(35 cards)
vocab - defintions for
*Recombinant DNA
*Genetic manipulation / genetic engineering
*Clone
*Gene cloning
*Recombinant DNA - DNA that has been formed artificially by combining two or more fragments from different sources.
*Genetic manipulation / genetic engineering - directed or pre-determined alteration of genotype of an organism.
*Clone - DNA molecule/cell/organism that is genetically identical to the DNA molecule/cell/organism from which it was derived.
*Gene cloning - is a biological method of purification. DNA from the gene of interest can be inserted into a “vector” (often a plasmid) which is then introduced into a host cell. When the vector replicates, the inserted DNA is also replicated, allowing large amounts of it to be isolated.
Outline the importance, and main experimental and commercial applications of gene manipulation technologies.
Analyse the cloned gene (sequencing, determination of expression profile etc.).
Where relevant, determine if gene is involved in inherited diseases-develop diagnostic tests, therapies
Produce recombinant protein and/or pure mRNA by in vitro transcription
Make specific mutations in the gene in vitro
Introduce into plants (generate transgenic plants)
Introduce into animals (generate transgenic animals)
Make precise changes in genes in vivo, e.g. using CRISPR
applications of genetic manipulation
A research tool:
Once a gene has been cloned, Its nucleotide sequence can be determined.
From this:
1. The amino acid sequence of the encoded protein can be deduced.
2. The evolutionary history of the gene and its host species can be analysed.
3. Primers for PCR can be designed.
The clone can be used as a probe, e.g. to determine in what tissues, and at what developmental stages, or disease states, the cloned gene is expressed.
Devise experiments to understand the function of genetic material or genes. This can include manipulating the organism from which it is derived so that the gene is either over-expressed, or mutated so as to be non-functional (reverse genetics).
Medicine:
Once a gene has been cloned:
Genes involved in genetic diseases, or in cancer,
disease-associated mutations within the gene can be
identified.
This allows:
*The molecular basis of the disease to be determined.
*The development of diagnostic tests, and sometimes new therapies (Some RNA or DNA based), for the disease.
*Techniques to rapidly sequence entire genomes allow:
(i) The genetic changes in cancers cells to be identified.
(ii) Foetal DNA present in the plasma of a pregnant woman to be analysed. (Basis of non-invasive pre-natal testing for chromosomal abnormalities).
biotechnology:
Pharmaceutically or commercially important protein can be expressed in a suitable host organism (e.g. E. coli or yeast) allowing large amounts of the protein to be manufactured
*DNA profiling in forensic science and paternity testing.
*Genetic manipulation of organisms (plants, animals, microbes)
*to give altered growth or disease resistance properties, improve nutritional value or enable bioremediation.
*to produce commercially important compounds or proteins
Describe the reactions catalysed by restriction enzymes and the DNA sequences those enzymes recognise.
Restriction enzymes were discovered because the growth of bacteriophages is restricted in some types of bacteria.
These bacteria make restriction enzymes that cleave
DNA from infecting bacteriophages
*Restriction enzymes recognise, and cleave at, specific
DNA sequences. These are commonly 4 or 6 base-pairs (bp) in length (Type II restriction enzymes)
DNA fragments produced by restriction enzyme digestion have either “sticky” or blunt ends
Restriction enzyme cutting sites are palindromic - the top and bottom strands have the same sequence (read 5’ to 3’)
Describe the use of DNA ligase in gene cloning.
Discuss the features of basic plasmid vectors used in gene cloning.
*DNA is cloned by inserting it into a vector – A DNA molecule that carries the exogenous DNA fragment into a host cell in which the vector (and the inserted DNA) can replicate.
*Modified plasmids are the most commonly used vectors
Host cells include:
E. coli (most routine cloning procedures)
Other types of bacterium
Yeast
Plasmids used as vectors in gene cloning must have:
*One or more unique restriction enzyme sites (i.e. only occurring once in the plasmid) into which DNA can be ligated. (Often designed to contain a multiple cloning site / polylinker region)
*An origin of replication specific to for the host organism.
*A selectable marker that allows cells with the plasmid to be distinguished from cells that lack it. Usually an antibiotic resistance gene.
Other desirable features of a plasmid vector:
*High copy number
*Small size (a few thousand bp)
*A means of distinguishing recombinant plasmids (i.e. with exogenous DNA inserted) from non-recombinant plasmids.
Isoschizomers - Enzymes which recognize same site and cuts in the same way
Neoschizomers - Enzymes which recognize same site but cuts differently (SmaI, XmaI)
cloning a gene of interest
Clones of genes may be obtained from gene libraries –These are large collections of cloned DNA fragments, representing all or most of the genes present in the organism from which the library has been prepared.
Or
If you know the sequence the gene of interest can be amplified from genomic DNA or cDNA by PCR (or chemically synthesised).
types of gene clone
Cloning of chromosomal DNA generates genomic clones.
*Genomic clones must be used if the promoter or the intron-exon structure is to be analysed since promoter and intron sequences are not present in cDNA .
*Genomic clones cannot be used to make protein in E. coli since it cannot splice out introns.
*Cloning cDNA (DNA complementary to mRNA)
generates cDNA clones.
*cDNA clones must be used if the protein made by the gene is to be produced by
recombinant bacteria.
productions of proteins in e.coli
*Protein that is otherwise difficult to obtain can be made in large quantities by expression in genetically manipulated organisms.
*A protein being manufactured by a genetically manipulated organism will usually represent >1% of all the protein made by the organism.
*E. coli are commonly used as to produce valuable proteins since they are easily manipulated and can be easily grown on a large scale.
*Other hosts for protein production include other bacteria, yeast, fungi, cultured mammalian cells, insect cells etc.
Difficulties that must be overcome when expressing eukaryotic genes in prokaryotes
problem:
Bacterial cells cannot remove introns from 1° transcripts.
Solution:
Obtain the eukaryotic gene from a cDNA library. cDNA clones are prepared by reverse
problem:
The promoters and terminators of eukaryotic genes do not function in prokaryotic cells.
Solution:
Insert the cDNA from the eukaryotic gene into an expression vector that contain a prokaryotic promoter and terminator either side of the inserted cDNA.
problem:
In order to be translated a prokaryotic mRNA needs a ribosome binding site.
Eukaryotic mRNAs do not have ribosome binding sites.
Solution:
Use an expression vector that also contains a ribosome binding site immediately before the site of cDNA insertion.
Explain the difference between cDNA and genomic DNA and describe preparation of double-stranded cDNA.
Discuss how bacteria may be manipulated in order to produce pharmaceutically or commercially important proteins.
Poly Chain Reaction(PCR)
PCR is the targeted amplification of a specific DNA
sequence. This may be a small fragment (e.g. a gene) present in a large population of DNA (e.g. a genome).
*The amount of a chosen DNA fragment can be
increased a billion-fold or more in 2-3 hours.
*For example, starting with human DNA from a few cells,
PCR could be used to amplify the b-globin gene until >10 copies of the gene are present
PCR may be used in:
Cloning of genes
Measurement of gene expression
DNA profiling
Diagnosis of genetic and pathogen-induced disease
PCR requirements
To amplify a gene of interest you must know the sequence (at least of the ends or flanking regions)
*Target DNA
*Primers – 2 short segments of single stranded DNA – bind to sequence to enable synthesis to start
*A heat stable DNA polymerase - Taq DNA polymerase from the bacterium Thermus aquaticus
*dNTPs - a mixture of dATP, dCTP, dGTP and dTTP
*Buffer containing Mg2+ (cofactor for polymerase)
*Thermal cycler – computer programmed heating block – rapidly changes temperature
PCR primers
*Oligonucleotides - usually 17-25 nucleotides.
*Two primers are needed – one to base-pair with each end of the DNA fragment being amplified.
*Primers make PCR specific - they determine which DNA fragment is amplified;
- must be of high specificity to reduce non-specific annealing
21nt oligonucleotide = 421 chance of occurring elsewhere
*Primers are present in huge excess compared to the fragment of DNA being amplified
Explain how a PCR reaction is assembled and discuss the importance of role of the primers and Taq DNA polymerase.
Explain the events that occur during the denaturation, primer annealing and DNA synthesis steps of a PCR cycle.
PCR usually involves 20-40 cycles of amplification. Each cycle has three steps.
Denaturation - Incubation at 95°C (typically 30-60s) to denature DNA into single strands
Annealing - Incubation at 40-65°C (typically 30s) to allow primers to anneal to ssDNA
Extension - Incubation at 72°C (typically 1-3 min). Optimum temperature for Taq DNA polymerase so DNA synthesis can occur.
Experiment requiring 30 PCR cycles takes ~2-3 hours.
PCR-polymerase
Polymerases have been continually developed and improved.
Versions of Taq DNA polymerase still widely used – cheap and efficient
*Problem = error prone ~285/106 bases mis-incorporated (E. coli DNA polymerase ~9/106)
Proofreading polymerases (e.g. Pfu Pyrococcus furiosus) - have RNAse H-like domain, therefore 3’- 5’ proofreading (also has longer half-life than Taq)
Reduces chance of errors, but more expensive
*Have to ensure dNTPs present prior to adding enzyme (Hot Start enzymes)
*(Occasionally we want error prone PCR)
role of PCR primers
Primers can be designed to incorporate non-specific sequences.
*Sufficient specificity to target sequence and optimized PCR conditions enable amplified product to be made.
Enables incorporation of;
*new restriction sites
*tags for purification
*mutations in amplified sequence
*flanking sequences to enable homologous recombination (gene replacement)
DNA synthesis for gene cloning
Recently become much more affordable, can have whole gene synthesised (~1.5 kbp) and cloned in a vector for ~£250 and delivered in less than 1 month.
Design DNA sequence exactly as you want it
*Exclude / include restriction sites
*Select codon usage (dependent on host to be used for expression)
*Can generate genes just from a sequence - no source DNA or cDNA required
Explain how to make mRNA (e.g. for vaccines) in vitro.
Many plasmid vectors (e.g. pGEM3Z) include promoters for bacteriophage
RNA polymerases to allow transcription of inserted DNA in vitro.
eg. Making RNA vaccine against SARS-Cov2:
Insert gene encoding virus spike protein into suitable plasmid vector
Transcribe in vitro, with uridine replaced by pseudouridine (reduces non-specific immune activation and increases stability).
Add 5’ cap and 3’ poly A tail to RNA
Remove vector DNA, purify spike protein mRNA
Package pure spike protein mRNA into lipid nanoparticles
VACCINE
Explain the basis of gel electrophoresis.
Agarose (polysaccharide, from seaweed) (0.5 - 2 % w/v agarose)
Seperates DNA and RNA molecules according to their size Enables
* Estimation of size of DNA fragments
* Monitoring of restriction enzyme cleavage
* Preparative isolation of cleaved DNA fragments
* Assessment of purity of DNA
* Investigation of conformations of DNA
- DNA (or RNA) is pipetted into wells made at one end of the gel.
- An electric field is applied - DNA and RNA migrate towards the positive electrode.
- Gel is porous - smaller fragments migrate faster than larger fragments.
Rate of migration of linear molecules is inversely proportional to the log10 of their size. - The gel is treated with a stain that allows individual DNA/RNA fragments to be seen as distinct bands on the gel.
Outline methods to detect specific DNA and/or RNA molecules
Intercalating dye used to visualize DNA with UV
* Ethidium Bromide (EtBr)
* SYBR Safe
DNA ladder (marker) enables estimation of fragment sizes
DNA profiling
- A Short Tandem Repeat (STR) is a sequence of 2-6p (e.g. TCAT) repeated from 1-50 times.
- DNA profiling relies on differences in STR repeats in individuals’ genomes.
- STRs occur at many sites in the genome of humans and other animals. e.g. an STR on chromosome 11.
- The number of copies of the repeat in a particular STR varies enormously between different individuals (and between the two alleles of the STR in the same individual).
- PCR using primers that recognise sites in the non-varying DNA either side of an STR. Different individuals generate a different pattern of PCR products.
- The size of the PCR products depends on the number of repeats.
– Each person’s DNA produces two PCR products from their two alleles
– The number of repeats varies between alleles and from person to person.
To increase the degree of discrimination, several STRs would usually be analysed from each person being tested.
