Recombinant DNA Technology PDF

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Summary

Recombinant DNA TechnologyOverview Genetic manipulation may start with the cloning of a DNA fragment of interest (most often a particular gene), though use of PCR may allow this to be circumvented  A clone is a DNA molecule/cell/organism that is genetically identical to the DNA molecule/cell/organ...

Description

Recombinant DNA Technology Overview    

Genetic manipulation may start with the cloning of a DNA fragment of interest (most often a particular gene), though use of PCR may allow this to be circumvented A clone is a 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

Stages 1. 2. 3. 4. 5. 6.

Obtain DNA from organism of interest Break into fragments Ligate into vector (place DNA fragment into plasmid) Introduce into bacterial cells (transformation) Identify bacteria with a plasmid carrying the gene of interest Grow cells and purify plasmid DNA

Forward Genetics 

Phenotype – gene

Reverse Genetics 

Gene – phenotype

A Research Tool 

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Its nucleotide sequence can be determined. From this: - The amino acid sequence of the encoded protein can be deduced - The evolutionary history of the gene and its host species can be analysed - Primers for PCR can be designed The clone can be used as a probe to determine in what tissues, and at what developmental stages, the cloned gene is expressed. Altered gene expression during disease can be detected The biological function of the gene can be tested by 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 

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Once a gene has been cloned, If the gene is involved in a genetic disease, or in cancer, the DNA sequence of the normal gene can be compared to the sequence of the same gene from a sufferer of the associated disease. This allows: - The underlying cause of the disease to be determined. - The development of diagnostic tests, and sometimes new therapies, for the disease. Techniques to rapidly sequence entire genomes allows: - The genetic changes in cancers cells to be identified

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Foetal DNA present in the plasma of a pregnant woman to be analysed - This is the basis of non-invasive pregnancy testing for chromosomal abnormalities

Biotechnology 

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Once a gene has been cloned, if the gene encodes a pharmaceutically or commercially important protein, it can be inserted into a suitable host organism (e.g. E. coli or yeast) and expressed allowing large amounts of the protein to be manufactured. Gene manipulation technology has also been exploited through the use of DNA profiling (DNA fingerprinting) in forensic science and paternity testing. The genome of an animal or plant can be altered to give altered growth or disease resistance properties, or to cause it to produce commercially important proteins.

Cutting DNA with Restriction Enzymes 

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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 DNA fragments produced by restriction enzyme digestion have either “sticky” ends or blunt ends Restriction enzyme cutting sites are palindromic – the top and bottom strands have the same sequence (read from 5′ to 3′) Restriction enzymes digest to leave a 3′-OH and 5′-phosphate

Restriction Enzymes  

Restriction enzymes: Eco RI is an example of an enzyme that recognises a 6 bp target site and cleaves DNA to leave sticky ends Restriction enzymes: Hae III is an example of an enzyme that recognises a 4 bp target site and cleaves DNA to leave blunt ends

Joining DNA with DNA Ligase  

DNA ligase can make covalent bonds between two different DNA fragments that have the same sticky end DNA ligase can also join blunt ended fragments together, but the efficiency is far lower than ligation of sticky ended fragments

Vectors and Hosts   

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 - Other types of bacterium - Yeast

Key Features of Plasmid Vectors 

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Plasmids used as vectors in gene cloning must have: - One or more unique (i.e. only occurring once in the plasmid) restriction enzyme sites into which DNA can be ligated - An origin of replication. - A selectable marker that allows cells with the plasmid to be distinguished from cells that lack it. This is usually an antibiotic resistance gene Other desirable features of a plasmid vector: - High copy number - A means of distinguishing recombinant plasmids (i.e. with exogenous DNA inserted) from non-recombinant plasmids

Cloning into plasmid vectors and distinguishing between recombinants and non-recombinants  

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Any cells that do not take up a plasmid molecule are killed by ampicillin X-gal is a synthetic substrate for b-galactosidase. It is cleaved by b-galactosidase to give a blue dye

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Cloning a Gene of Interest  

DNA of a specific gene can sometimes be obtained by PCR (see next lecture) 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

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 But, genomic clones cannot be used to make protein in E. coli since E. coli 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

Making CDNA 

The mRNA used must come from a tissue that expresses the gene being cloned – e.g. to clone the insulin gene, pancreas mRNA must be used – liver mRNA would be no good since liver does not make insulin

Production of pharmaceutically or commercially important proteins    

Protein that are 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 (and sometimes other bacteria) 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 yeast and cultured mammalian cells

Difficulties that must be overcome to express Eukaryotic genes in E. coli cells 

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Eukaryotic genes containintrons. Bacterial cells cannotremove introns from 1° transcripts - Obtain the eukaryotic gene froma cDNA library. cDNA clones are prepared by reverse transcription of mRNA and so lack introns The promoters and terminators of eukaryotic genes do not function in prokaryotic cells - Insert the cDNA from the eukaryotic gene into an expression vector that contain a prokaryotic promoter and terminator either side of the inserted cDNA In order to be translated a prokaryotic mRNA needs a ribosome binding site. Eukaryotic mRNAs do not have ribosome binding sites - Use an expression vector that also contains a ribosome binding site immediately before the site of cDNA insertion

DNA (or RNA) fragments of differing size can be separated using gel electrophoresis    

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Gels are slabs of agarose or polyacrylamide 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 The gel is porous and acts like a sieve - smaller fragments migrate faster than larger fragments. The rate of migration of linear molecules is inversely proportional to the log10 of their size Following electrophoresis the gel is treated with a stain that allows individual DNA/RNA fragments to be seen as distinct bands on the gel

Sanger Sequencing    

Used to sequence a single gene or DNA fragment “Sanger” Sequencing uses dideoxynucleoside triphosphates and DNA polymerase DNA synthesis terminates since no 3′-OH available Sequencing uses ddATP, ddCTP, ddGTP and ddTTP, each labelled with a different fluorescent dye

Next Generation Sequencing 

Used to sequence a whole genome, or thousands or millions of DNA fragments

Detecting Specific Nucleic Acid Sequences   

Specific mRNA (or DNA) molecules in a mixture can be identified by using a probe that can base-pair (hybridise) with them The probe hybridises only to the complementary fragment Blotting” methods are used to detect specific molecules present in a mixture that has fractionated by electrophoresis

Determining when and where a gene is expressed 

PCR

In situ hybridisation

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Targeted amplification of a specific DNA sequence Those may be a small fragment in a large population of DNA e.g. genome Can increase the copies of DNA by a billion fold or more in 2-3 hours Used in: - Cloning of genes - Measurement of gene expression - DNA profiling - Diagnosis of genetic and pathogenic diseases

Requirements for PCR 

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Primer - Oligonucleotides – short (17-25 nucleotides) freagments of single stranded DNA that are chmically synthesised 2 primers are needed – one to base pair with each end of the DNA fragment The primer make PCR specific – they deterimine which fragment is amplified A heat stable DNA polymerase – usually Taq DNA polymerase

Primers  

The forward primer directs synthesis of the top strand The reverse primer directs synthesis of the bottom strand

Performing a PCR Reaction 

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A PCR reaction contains: 1. A small amount of DNA being amplified 2. Primers 3. A mixture of dATP, dCTP, dGTP and dTTP 4. Taq polymerase 5. Micture is placed in a thermal cycler Incubation at 95 degress to separate the template DNA Cool to 45-65 degrees to allow primers to anneal to the single stranded DNA Incubation at 72 degrees – optimum temperature for Taq DNA to synthesis DNA strand Each step typically takes 30-60 seconds, an amplification experiment requiring 30 PCR cycles takes 2-3 hours

Primers Can Bind Within a DNA Fragment 

Only the sequence between and including the binding sites for the primers

DNA Profiling     

Allows individuals to be identifies Relies on differences between the genome of different induvial Based on differences in short repeated sequences known as short tandem repeats (STRs) A sequence of 2-6p repeated 1-50 times STRs occur at many sites in the genome of humans and other animals

Obtaining a DNA Profile

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To generate a DNA profile, PCR is carried out using primers that recognise sites in the nonvarying DNA either side of an STR The size of the PCR fragment depends on the number of repeats Each person’s DNA produces 2 PCR products from their 2 alleles with the number of repeats varying between the alleles

Paternity Testing 

For each STR a child inherits one allele, one from each parent

Huntington’s disease      

Late onset disease Autosomal dominant genetic disorder Causes progressive neurological deterioration The mutation that causes the disease is a trinucleotide expansion PCR is performed using primers that flank the trinucleotide expansion The size of the PCR product depends on the number of CAG repeats that are present

PCR in the diagnosis of genetic disorders 

Most other genetic disorders, e.g. Cystic fibrosis and sickle cell disease, can also be diagnosed using PCR-based tests

Detection of Pathogens  

Infections by certain pathogens can be detected by PCR It is done by using primers that recognise

Genetic Manipulation Transgenic Animal  

An animal that has exogenous DNA inserted into its genome Can be made to: - Produce pharmaceutically/commercially important proteins by generating transgenic animals that secrete the protein into their milk - Improve farm animals

Transgenics animals in aquaculture – ‘AquaAdvantage salmon’ Advantages:  

Increased growth rate (but not ultimately larger than normal). Improved utilisation of diet

Concerns:  

Human health - altered levels of hormones/allergens? Environment - escaped transgenic fish may breed with wild population

1. An exampling of ‘pharming’ – production of valuable proteins in transgenic animals Transgenic Plants

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Transgenic plants (plants that have exogenous DNA inserted into their genome) are generated with the aim of:

1. Manufacturing commercially important proteins. 2. Improving” agricultural species, for example by: - Producing plants that are resistant to pests (e.g. insects). - Producing plants that are resistant to herbicides. - Producing plants with improved nutritional properties The Ti Plasmid – a tool for making transgenic plants 

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Infection of some plants by the bacterium Agrobacterium tumefaciens causes plant tumours (known as crown galls). Tumours arise because Agrobacterium contains the Ti (tumour inducing) plasmid The Ti plasmid contains a region of DNA (the T region) that promotes tumour formation. During an infection by Agrobacterium the T region becomes integrated into the genome of the infected plant cells The Ti plasmid can be used as a vector to introduce exogenous DNA into plants susceptible to infection by Agrobacterium.

Using CRISPR to manipulate genomes    

An adaptive immune system from bacteria and archaea! During a bacteriophage infection, small fragments of bacteriophage DNA become inserted into the bacterial genome at the CRISPR locus Transcription of these fragments produces CRISPR RNA (crRNA). This forms a complex with a trans-acting RNA (tracrRNA) and a DNAse enzyme (Cas9). If the bacterium is subsequently infected by the same type of bacteriophage, the Cas9/ tracrRNA/crRNA complex recognises the bacteriophage DNA (due to base pairing with crRNA) and cleaves it

So CRISPR can cleave a target-gene in vivo?  

Following cleavage by CRISPR, DNA repair may lead to inactivation of the target gene, or can be exploited to make a precise change (edit) in the target gene Cleavage of a target gene by CRISPR leads to inactivation of the gene or, if a template for homology directed repair is provided, precise alteration (editing) of the gene...