Lac Operon And Catabolite Repression
Lac Operon -> can utilise glucose and lactose. This is Diauxic growth.
Catabolite Repression -> Crp-cAMP complex binds to promotor. High glucose levels inhibit cAMP. Must be present for RNA polymerase to bind.
The trp Operon
Repressor switched off when no tryptophan bound. This is a biosynthetic operon.
Attenuation = where leader region has 4 C-G rich regions followed by A-T rich region. Terminator only forms when tryptophanyl-tRNA in short supply. Pauser forms after regions 1 & 2 while translation starts. Progress of ribosome destroys pauser.
If NO tryptophanyl-tRNA:
Ribosome forced to wait at UGC at region 1. When region 3 is transcribed it base pairs to region 2 forming an antiterminator. Transcription continues through whole operon.
Ribosome synthesises whole operon and pauses at stop. When region 3 & 4 transcribed, they bind together. The stops transcription/translation and the protein formed will be degraded.
Eukaryotes vs Prokaryotes
Transport of stable RNA from nucleus to cytoplasm
Transcription in nucleus and translation in cytoplasm
Some proteins transported into mitochondria, chloroplasts, vacuoles
No nucleus. RNA is unstable
Transcription and tralslation simultaneous
No such structures as mitochondria, chloroplasts, vacuoles
RNA polymerase 1,2 & 3.
RNA Polymerase 1 transcribes most rRNA
RNA Polymerase 2 transcribes all mRNA
RNA Polymerase 3 transcribes tRNA and one small rRNA.
All 3 complexes are about 10 protiens but type 2 is the most important in terms of expression
Promotor sequences = CAAT box & TATA box. There is no known simple terminator and genes contain introns and occur singularly rather than in an operon. mRNA is capped at 5' end (Methyl guanosine) and tailed at 3' end (polyadenosine).
Prokaryotes don't have most compartments or these features. Phosphorylation, methylation and glycosylation are more limited.
Capping and Tailing
With the mRNA cap, the first couple of nucleotides in original mRNA have methyl groups.
There is no known terminator of transcription so it continues beyond the end of the gene. mRNA is then cut and couple of hundren adenosines are added.
mRNA without the cap translates poorley. mRNA without the tail doesn't translate at all.
Polyadenylate-binding protein (PADP) binds to polyA tail. The ribosome can't bind to the cap without initiation factors and some of these need PADP.
(Genes transcribed by RNA polymerase1 & 2 don't encode proteins so have a different mechanism of termination. They have specific terminator sequences.)
Introns: non-coding sequences in the gene that appear in the mature mRNA even though they interrupt the coding sequence. (Peices of codon between the introns are called extrons).
Occurance of Introns is variable
May be small (70-80nucleotides) or large (103-106nucleotides)
Average human gene = 27,000 bp with 9 exons. Introns make up 24% human genome, exons make up 1%.
Sequence always starts with GT and ends with AG
Spliceosome consists of severnal RNAs and proteins and removes introns.
Genes can be cut in many different ways depending on which intron boundary is spliced.
Introns occur that the splicing doesn't need to be too exact.
Leghaemoglobing in legumes and Haemoglobin in humans have the same introns at the same points.
Both prokaryotes and eukaryotes have a pair of conserved (consensus) sequences. Prokaryotes = Pribnow box (10bp). Eukaryotes = TATA box (30bp).
However, spacing of the sequences and transcription start point are different in both types of organism. The prokaryote -35 is highly conserved, the eukaryote -80 may take many forms.
The eukaryotic core promotor is where RNA polymerase II binds, after binding to transcription factors. TF2D (15 proteins but only 1) binds to TATA box and TF2B binds upstream.
The promiximal promotor is outside the region where DNA polymerase binds. The CAAT box is a typical poroximal promotor binds to transcription factors, the DNA loops round and these are brought into contact with DNA polymerase.
The distal promotor isn't very well defined. Other control factors such as silencers, enhancers and insulators bind here.
SV40 enhancer is essential for transcription. It consists of a repeated 72bp sequence. It works just as well turned 180 degrees and increased expression of the genes near it.
Expression of foreign genes
Why express foreign genes? To alter the properties of crops, animal stock or microorganisms. It can make useful proteins from animal/bacteria/plant stock or alter biochemical pathways. It can also be used to make safe and effective vaccines.
To insert the genes, chemical treatment such as Ca2+ or electroporation could be used. You could also use a pathogen (virus or bacterium) or inject into the nucleus.
Using a vector or attaching a replication origin will help maintain the DNA in the cell.
To be able to tell which cells are recombined, add a gene that gives resistance to antibiotics such as penicillin.
To switch on the gene, and control it, enhancers, oporator and upstream activating sequences should be included.
To ensure the protein product is correctly produced you must choose a suitable host organism and tissue type.
Production of Human Somatotrophin in E. coli
Obtain the gene by letting the cell transcribe the mRNA and splicing out the introns. Then make a DNA copy - cDNA.
We must remove the polyA tail, add a prokaryotic promotor and terminator.
The DNA is cut with enzyme C . The cDNA fragment and the artificial fragment were then joined at the site for enzyme C by DNA ligase.
The promotor, control sequences and terminator are all joined by using a cassette. This cassette is inserted into an E.coli vector with an origin of replication, and an ampicillin resistance gene.
The joined up plasmid is inserted into the bacterium by soaking the bacteria in calcium chloride solution for one hour, mixing them with the DNA and heating breifly to 42 degrees C.
This can now be used to treat patients.
Production of vaccine for foot and mouth disease v
Production in the past was difficult because the heat inactivated vaccine could be heated too much so it didn't work, or not enough that it was still pathogenic.
Antigenic material consists of 2 coat proteins VP1 and VP3. If the gene encoding these could be cloned and expressed in E. coli the protein produced could be used as a vaccine.
cDNA can be made of the gene, and it has no introns to worry about. This was put into a fusion cassette which has the restriction endonuclease after the ATG codon. VP3 can be used in this way.
Production of Bovine luteinising hormone in cultur
LH is a member of the glycoprotein hormone family. Each member has the same alpha unit, a different beta unit, both of which are poly peptide units.
The two subunits are put in an expression cassett with SV40 promotor region and a Poly A tail.
The cassett also contains a mouse dhfr gene to give resistance to methotrexate.
Production of glycoprotein hormone in plant cells
Erythropoeitin (EPO) has haematopoetic activity and tissue protective activity. Recombinant human EPO is used to treat anemia.
It could be used to protect tissue but is toxic in high dosage due to the sialic acid side chain. This is sometimes treated enzymatically to remove the sialic acid so it can be used for tissue protection, but this is expensive.
Genetic improvement of livestock
Domestication - Galton's Conditions
They should breed freely
They should be hardy
They should have an inborn liking for man
They should be comfort loving
They should be found useful by savages
They should be easy to tend
Animal genetics = Study of inheritence in animals
Animal Breeding = Applications of principles of animal genetics. The goal is improvment
No Animal is best for all situations.
Traits of economic importance in farm livestock
Most traits of economic importance are controlled by many different loci and don't usually have discrete categories.
Main principles: Alleles are paired in parents but pass to offspring singly and when animals produce sperm/eggs the two alleles will be represented equally.
We need to know how many animals there are of each genotype to get the genotype frequency
Hardy-Weinber equilibium -> Gene frequency remains the same if there is no;
- Selection for or against particular alleles at a locus
- Movement of animals into/out of large random mating populations of animals
However, this is rare in nature due to genetic drift. Genetic improvement depends on genetic variation, this is the most important component. Sources of genetic variation:
- Differences in gene frequency
- Segregation of genes (Alleles)
Selection between and within breeds
Demand for lower cost, higher quality product and competition from imported products fuel a need for genetic improvment.
Genetic improvment is valuable because it is permenant, accumulative and highly cost-effective.
Group breeding schemes started because farmers wern't happy with stock produced. They formed nucleus flocks or herds and used in depth recording and selection to bring about genetic improvment.
Selection between breeds causes a rapid and dramatic change when there are large genetic differences. It can only be achieved once and is very costly so in practice, gradual selection is practiced.
Breeds should be compared in the same 'relative' environment and at the same end-point. You should also sample properly by using a large number and randomly sampling.
Genotypes don't always rank the same in different environments. This is called:
- Genotype x environmental interaction OR
- Breed x production system interaction
Selection between and within breeds cont.
Selection within breeds is comparison of animals of the same breed and mating the preferred animals to produce the next generation to achieve continuous genetic change accross generations. However, genetic variation must be present!
Requirements for within-breed selection
- Breeding Goal
- Selection Criteria
- Designing and implementing the breeding program
- Monitoring progress
TANDEM SELECTION => Selection for one train for one generation followed by a different train for one or more generations
INDEPENDANT CULLING LEVELS =>More consistent, all animals must qualify in standard level in each trait of importance.
SELECTION INDEX => Optimum method - source of overall gentic merit for each animal based on their own and/or their relatives performace. The animal can compensate for one trait by excelling in another.
Selection between and within breeds cont..
Selection between breeds, selection within breeds and the first four applications of crossbreeding all exploit additive genetic merit. Heterosis is a result of non-additive gene action i.e. dominance or epistasis,
Heterosis is greatest for traits asoociated with reproduction, survival and fitness. Also for breeds that are genetically diverse.
Crossbreeding just to exploit heterosis is only justified if the crossbreed is better than the best parent breed.
INDIVIDUAL HETEROSIS: Influencing performance as a result of animals themselves being crossbred
MATERNAL HETEROSIS: Influencing the reproductive and other maternal performance characteristics of crossbred females
PATERNAL HETEROSIS: Influencing the reproductive performance of crossbred males
Factors affecting rates of genetic improvement
Response to selection => The change in mean performance in a population of animals as a result of selection, usually per year or generation. Three factors affect the response to selection.
- 1) Selection differential - s (selection intensity - i). The difference between the mean performance of selected animals and the overall mean performance of the group of animals from which they were selected.
Unequal numbers of each sex are usually selected but each sex contributes half of their genes to the offspring so selection differentials must be calculated sperately for each sex before taking an average to get the overall selection differential.
S is dependant on the variation in performance for the trait of interest. The wider the variation in performance, the higher the selection differential that can be acheived. Also, the fewer top animals selected, the higher the selection differential.
- 2) Heritability - h2 (genetic variation). The proportion of the superiority of parents in a trait which, on average, is passed onto offspring.
To calculate heritability is to select parents on this trait, measure the selection differential achieved, mate them, measure the same trait in the offspring and then calculate (using regression) how much superiority of parents was passed onto the offspring.
Factors affecting rates of genetic improvement con
- 3) Generation interval - L. The average age of parents when their offspring are born.
- unequal numbers of male and female parents
- often breed at different ages
- so therefore, generation intervals have to be calculated seperatly for each sex and then averaged.
- Response to selection (R) = Selection differetial (S) x Heritability (H2)
- Response to selection per year = Selection differential x heritability Generation interval
To predict selection differential before starting breeding, we can use a normal distribution (Gaussian) curve. The superiority of animals selected, as expressed in SD units, is called the standardised selection differential or selection intensity. Fewer animals selected, higher the selection intensity. S = i x SDp (phenotypic standard deviation).
Annual response to selection is highest when:
- Selection intensity and heritability are high
- Generation interval is low
Use of information from relatives
Estimated breeding value - EBV
Using information from animals ancestors allows you to do pedigree selection. Information combined of animals ancestors goes into a pedigree index.
- Selection on the animals own performance is the simplest method of selection and most favoured historically. It has potentially high accuracy and works best for triats of high h2.
- Sib selection/sib test is useful if the trait of interest is only measurable in one sex. However, this has low accuracy due to low numbers.
- Progeny testing is often used for milk production selection. This has high accuracy and is useful for sex limited traits and carcass traits.
Proportion of genes in common with other relatives are only averages since recombination and segregation lead to change in variation. Each generation that seperates two relatives roughly halves the genes they have in common.
Change R = (i x h2 x sdp)/L to R=(i x h x sda)/L or even R = (i x r x sda)/L Where r = accuracy of selection and sda = additive genetic standard deviation.
Use of information from relatives cont.
Accuracy of selection (r) depends on three things:
- The heritability of the trait concerned
- The source of information e.g. class of relative
- The amount of information available from relatives
Accuracy is a correlation between measured traits and true breeding value (TBV). Accuracy of selection of a single record of performance is the product of genes they have in common and the square root of heritability (r=h). If the selection is on a single measurement from one parent/offspring accuracy is halved.
Repeated records can give additional clues to the breeding value of an animal and enhance the accuracy of selection. Any difference in these performances if due to permenant environmental effects. The value depends on repeatability.
P = (Ga + Gna) + (Ep + Et)
Repeatability sets an upper limit to heritability. It is concerned with the genetic part of the equation where as repeatability includes environmental aspects.
'Mating of related individuals' or 'mating of individuals more closely related than average for the population'.
Related animals have more genes (alleles) in common than unrelated animals. As well has having more favourable genes in common, they also have more unfavourable genes in common.
Inbreeding leads to serious genetic defects. Inbreeding depression is the manifestation of poor gene combinations that significantly decreases performance. Increasing homozygosity reduces the amount of genetic variation, reduces the response to selection and can lead to a decline in performance in traits.
Inbreeding coefficient (F) - 'The probability that both alleles at a single locus are identical by decent' Fx = (SUM OF)(1/2)^n(1+Fa) n = number of individuals in a path
- Rate of inbreeding per generation (DELTA F) = 1/8m + 1/8f
- Rate of inbreeding per year (DELTA F) = 1/8mL2 + 1/8fL2 L=average generation interval
Reproductive technologies include:
- Multiple Ovulation and Embryo Transfer (MOET)
Application of Reproductive Technologies
Advanced reproductive technologies can be used to overcome biological contraints. Strategies used to increase reproductive rate are: Genetic selection, Immunology and pharmacology and Embryo transfer.
Sire Referencing Schemes - a team of high genetic merit sires are used across a range of flocks by AI. Therefore, a common genetic baseline can be established in each flock.
Analysis is carried out by the BLUP (Best linear unbiased predictor) statistical procedure. It produces EBVs for each animal.
MOET increases selection intensity and rate of genetic gain while decreasing generation interval.
Cloning can conserve genetic resource while aiding genetic improvment, dissemination and modification.
Genetic modification can be used for:
- Gene function research
- Pharma - or Nutraceuticals
- Disease resistance
Traits => varients of a phenotypic character of an organism that may be inherited, determined by the environment or both are are visible.
Drift => changes in allele frequency via random sampling
Polymorphic loci => any locus that has more than one allele present within a population
QTLs => Quantitive trait loci
Genetics is the study of heredity. Genes are thread-like double helical molecules of DNA. Genes are the functional units of chromosomes.
Genetic variation is a result of genetic mutation, gene recombination and genetic drift. This is important to maintain biodiversity, to effect natural selection and so the pool of genes pass to the next generation.
Genetic variation can be from inter/intra specific variation naturally, or induced through mutagents, genetic engineering or polyploidy.
Divergence of populations can be due to drift, or natural selection leading to them being locally adaptive.
Genetic variation cont.
Intra-specific genetic variation refers to the range of genetic information available among all individuals of a species. (can be within-population or between-population).
Inter-specific variation refers to the range of genetic information available of related species that don't normally sexually reproduce with each other.
Polyploidy is the increase of complete sets of chromosomes, usually induced with chemicals.
Genetic engineering is the manipulation of the DNA of organisms to change inheritance or the in vitro manufacture of gene products.
Measurement is by looking at the phenotype (proportion of polymorphic loci and heterozygosity) and by protein electrophoresis. Quantification is through statistics (ANOVA).
Protein electrophoresis may miss silent or synonymous (changes that don't affect the amino acid) mutations.
DNA sequence variations can be detected by gel electrophoresis or restriction enzymes. Different patterns on the gel are called Restriction Fragment Length Polymorphisms (RFLPs).
DNA polymerase => an enzyme catalysing synthesis or breakdown of DNA.
Types of mutation:
- Point mutation
- Small insertions/deletions
- Gene duplication
- Major chromosomal change
Source of mutation:
- DNA polymerase errors during DNA replication
- External effects = chemicals, radiation
- Failure of repair mechanisms
However, a low level of mutational change is highly desirable to provide the ability for species to adapt to changing environments.
Physical mutagens = xrays, UV light
Chemical mutagens = Base analogues, Base modification, intercalating agents
Somatic mutations occur is somatic cells and cannot be inherited.
Germ-line mutations occur in the germ-line of a sexually reproducing organism and is inherited.
Mutation rate => The probability of a particular kind of mutation as a function of time
Mutation frequency => The number of occurances of a particular kind of mutation. It is expressed as a proportion of cells or individuals in a population.
bp substitions = Transistions : replacement with the same chemical group. Tranversions = replacement with a different chemical group.
Frameshift mutations can result in a readthrough of stop codons resulting in a longer protein.
Most mutations are spontaneous. All point mutations are spontaneous. Many of these are repaired and don't become fixed in the DNA.
Induced mutations can be caused by x-rays that ionise chromosomes, or by UV radiation that causes the formation of abnormal chemical bonds.
Chemical mutagens may occur naturally or be synthetic.
Base analogues are similar to normal nitrogen bases and are incorporated into DNA redily. Once in the DNA, a shift in the analogs form will cause incorrect base pairing during replication, leading to mutations.
Base modifying agents work by modifying chemical structure and properties of bases. Nitrogenouse acid acts as a deaminating agent, convertine C to U.
Advantages of using this (EMS) to mutate organisms is that it isn't volatile so is less likely to be breathed in. It can also be left over night to become completly inactive and is soluable in water.
Intercalating agents can be inserted between adjacent bases in dsDNA. It can alter the spacing of s-p backbone of DNA, often causing deletions and associated frameshift mutations and they don't cause base replacement.
This can upset DNA polymerase which sticks at a random nucleotide or jumps and affects its fidelity.
DNA & Genome sequencing definitions
- DNA sequencing : figuring out the order of DNA bases or nucleotides in a piece of DNA
- High resolution electrophoresis : the motion of dispersed particles relative to a fluid under the influence of a uniform electric field
- Methyl interference assay : analytic method to identify nucleotides that are important for DNA binding
- Primer : (Oligonucleotide) a strange of nucleicacid that serves as a starting point for DNA synthesis
- Klenow fragment : a large protein fragment produced when DNA polymerase I is cut by the protease subtilisin enzyme
- Polyacrilamide gel electrophoresis : (PAGE) a tecnique used to seperate DNA or protein molecules
- Autoradiography : an image on an x-ray film produced by the pattern of decay emmitions of a radioactive substance
- Genome sequencing : the determination of the order of the bases in the DNA of a genome
- Non-protein coding sequences : all the sequences is a genome that don't contribute to make a protein, including regulatory regions and pseudogenes
- Genome map : the set of landmarks in a genome
DNA & Genome sequencing
Two procedures involved:
- 1) Selective termination of DNA synthesis at specific nucleotides
- 2) High resolution electrophoresis
Sequencing methods include:
- Maxam and Gilbert method
- Sanger dideoxy method
Maxam Gilbert method is based on a chemical modification of DNA and subsequent cleavage at specific bases. It is technically complex, uses hazardous chemicals and is difficult to scale it up. The fragment in the 4 reactions are electropheresed in a denaturing acrylamide gel for size separation. The gel is exposed to an x-ray film for autoradiography.
The Sanger dideoxy method was developed by Frederick Sanger. It is more efficient and uses fewer toxic chemicals and lower amounts of radioactivity. It is based on the use of dideoxynucleotide triphosphates as DNA chain terminators. It uses a defined primer that binds to the 3' end of the template strand to be sequenced.
Sanger Dideoxy method
- The primer must be tagged at it's 5' end with a radioactive phosphate gel or flourescent dye
- Synthesis of a new strand of DNA is initiated from the primer and a chain termination process is used to stop synthesis selectivly at any one of the four DNA nucleotides
- This is performed by incorporation of a 2'-3'-dideoxy-nucleotide which leaves no 3'-OH group for further growth
- A series of fragments with various lengths will be generated which can be used to read a nucleotide sequence
- Four identical reaction mixtures are set up with : -Same template, -Labelled primer, -DNA polymerase, -DATP, DTTP, DCTP, DGTP, -One of these 4 => ddATP, ddGTP, ddTTP, ddCTP.
- Klenow fragment of DNA polymerase I supports 5' to 3' synthesis of DNA and it's often used for sequencing
- Synthesis of the new strands continues until a dideoxyribonucleotide is incorporated
- The new DNA strands in each reaction mixture are seperated by electrophoresis
- A polyacrylaminde gel seperates strands differing by as little as one nucleotide in length
- Aytoradiography is used to detect radioactive bands
- The sequence of the original template strand can be detected form the bands on the autoradiograph.
DNA & Genome sequencing cont.
High-throughput sequencing technologies are intended to lower the cost of DNA sequencing beyond whst is possible with standard dye-terminator methods.
After sequencing we need to know how the gene is organised, the structure, the function, how genes are related, how various parts are coordinated and other unidentified secrets.
To sequence a genome you break it up and sequence the pieces. You then reassemble it in the correct order.
Non-protein coding sequences make up a small fraction of DNA in prokaryotes. This all increases in eukaryotes.
A genome map is one dimensional and can include genes, promotors and regulatory sites. A map can help in the sequencing of a genome to understand the sequence.
Maps are used to:
- position where a clone came from
- help find other important parts of the genome
- identify clusters of related genes
- compare genomes of different species
Restriction endonucleases & cutting of DNA
Genetic enginering enables us to take genes out and anaylse them. The DNA can then be altered or re-inserted into an organism.
DNA can be cut by:
- Shaking the DNA violently causing random breakages
- Using specific targetted enzymes - endonuclease or exonuclease
Exonuclease => cleaves nucleotides one at a time from the end (exo) of a polunucleotide chain
HindII is the first restriction enzyme discovered accidentally in 1970. The bacterium Haemophilus influenzae can break down DNA from the phage. This discovery led to the development of recombinant DNA technology and this allowed large scale production of products such as human insulin using E.coli.
Applications of exonucleases:
- Removal of oligonucleotides before a PCR reaction
- Removal of chromasomal DNA in plasmid preparations
- Removal of DNA in RNA preparations
- Generation of ssDNA from linear dsDNA
Restriction endonucleases & cutting of DNA cont.
Recombinant DNA technology => The creation of a new combination of DNA segments that arn't found together naturally
Applications include basic research on control gene expression, forensic medicine and biotechnology.
Restriction endonucleases cleave the phosphodiester bonds of nucleic acids at an internal site. They are highly specific and can recognise 4bp (HaeIII), 6bp (EcoRI), 8bp (NotI) and more. The restriction sites that are recognised are usually pallindromes.
Frequency of enzyme recognition depends on the number of nucleotides used in the site of recognition and the expected frequency in the length of DNA.
Blunt ends are generated by shearing DNA or by cutting with blunt end endonucleases.
Overhang is a strech of unpaired nucleotides on the end of a DNA molecule, on either strand.
Sticky ends form transient double helical structures which can be joined together by ligase. If different DNA molecules are mixed, ligase will generate new combinations.
Restriction maps show positions of restriction sites in a DNA sequence.
Cloning of DNA
DNA cloning => a technique involving the insertion of a foreign DNA fragment into a vector capable of replicating seperatly in a host. Growing the host cell allows the production of multiple copies of identical inseted DNA.
Methods that have been used for DNA include:
- Cloning by restriction enzyme digestion and ligation of compatible ends
- T-A cloning directly from a PCR product
- TOPO-attached unidirectional cloning
- Recombination based cloning
Requirements for DNA cloning include:
- Foreign DNA such as a PCR product, genomic DNA or complimentary DNA.
- Host organism, could be bacteria, eukaryotic, mammalia/plant cells or insect cells
- Vector DNA that functions as a molecular carrier and facilitates its replication
- The foreign DNA must be ligated into the linearised vector. You need 2+ fragments of DNA, a buffer containing ATP and a T4 DNA ligase
- Bacteria cells take up naked DNA molecules. The cells need to be made competant so are heat-shocked. This allows the DNA molecules to get inside the cells. (Electroporating could also be used)
Cloning of DNA cont.
cDNA (complementary DNA) is synthesised from an mRNA by reverse transcriptase and DNA polymerase.
The membrane of cells are permeablised using an electric field. The DNA present can penetrate the membranes after the electric shock. The protocols differ for various species so the efficiencies of transformation ranges from 109 per ug DNA to 106 per ug DNA.
Conjugation: the natural transmission from donor to recipient. It occurs with a host cell that is not readily transformed. It involves formation of cell to cell junctions.
Transfection of DNA: DNA is packaged into phage particles in vitro. The phages are allowed to infect bacterial cells and is transiently expressed.
Selectable marker: A gene introduced into a cell that confers a trait for artificial selection
Hosts are grown in selective conditions to allow the identification of positive clones containing the carrier vector with the DNA of interest. These are:
- 1) Antibiotics 4) Nutrient requirements
- 2) Plaque type 5) Blue-white selection
- 3) Specific (hybridisation)
Cloning of DNA cont..
Once colonies are identified they are cultured in broth to increase numbers and therefore amount of DNA. Samples are also prepared for storage at -80. They can be kept for many years.
How is a segment cloned:
1) A segment of DNA needs to be isolated
2) In some instances restriction enzyme sites need to be incorporated into the DNA of interest
3) The DNA segment is digested with restriction enzymes
4) A vector is digested with the same restriction enzymes used to digest the desired DNA
5) The segment of DNA is attached to the DNA with ligase
6) The generalised plasmid is introduced into a bacteria to make more copies
- To isolate DNA
- To establish collections
- To perform further molecular studies
Artificial chromosome: a functional chromosome created by genetic engineering with a centromere, telomere and is transmi**ible in cell division
DNA libraries: a collection of fragmented DNA that is stored and propagated in a populationof microorganisms via molecular cloning
F-Factor: fertility factor in bacteria, also called episome and involved in DNA transduction
There are several types of artificial chromosome - YAC, BAC and Bacteriophage
Bacteriophages are viruses that infect bacteria. There sequence is known and is around 50Kb. They are linear ds molecules with ** complentary ends (cos region). They have cohesive termini.
Types of DNA vector include:
- Cosmids = contains DNA from bacteriophage lambda
- Fosmids = based of F-factor
- Plasmids = clones small segments of DNA
Gateway technology was invented in 1991 by Invitrogen. It is fast and flexible and highly efficient. it does maintain the oreintation and reading frame without using restrictioon enzymes or ligation. It can clone up to 4 segments at once.
Modern vectors cont.
Plasmids are small circular dsDNA that autonomously replicate independantly of the chromosome of the host cell. They are 'molecular parasites'. They carry one or more genes, some confer resistance to antibiotics. They have an origin of replication which is a region of DNA that allows multiplication of the plasmid within the host.
Properties of good plasmids are
- They must be small in size
- Their DNA sequence must be known
- They must produde a high number of copies
- They must have a selectable marker
- THey must posses a second selectable gene
- They must have a large number of unique restriction sites
Multiple clone site: short sequence of DNA with restriction sites, usually in a unique vector
Characteristics of pBR322:
- Medium in size - 4.4Kb AmpR allows selection
- Doesn't have multiple clone site ORI ensures plasmid is copied
- In the muddle of the tetracycline resistance gene Two replica plates used
DNA polymerase: an enzyme that's involved in polymerisation of deoxyribosenucleotides into DNA. The new synthesised strand is complementary to the template strand.
Annealing: is the pairing of DNA or RNA by hydrogen bonds to a complementary sequence to form a double-stranded polynucleotide
TaqMan probes: are hydrolysed probes designed to increase the specifity of real time PCR assays
PCR is a method of producing millions of copies of a target sequence from template DNA in a few hours, compared with bacterial cloning which takes much longer and is less efficient.
- DNA cloning
- Generation of hybridisation probes
- DNA sequencing
- Isolation of DNA for recombinant technologies
- Rapid screening of colonies
- Genetic fingerprinting
- Paternity testing or evolution relationships
PCR reaction requires:
- 1. dsDNA template
- 2. A heat-resistant DNA polymerase
- 3. Four nucleotides :dATP, dTTP, dCTP, dGTP
- 4. Two short ssDNA molecules serving as primers and complementary to each one of the strands of DNA
- 5. Magnesium ions
- 6. Buffer containing salt
Heat (94-99degrees) to denature DNA strands. Cool (50-65degrees) to anneal primers to template. Warm (68-72degrees) to activate Taq polymerase. REPEAT.
Taq polymerase extends primers to replicate DNA. The exact length target product is made in the 3rd cycle. It is heat resistant and is fast but has a low replication fidelity.
Pfu polymerase is heat resistant, has proof reading activity but is slow. By mixing this and Taq you can gain speed and fidelity.
Primers should be >15bps long and have a G-C content between 40-60%. They also need to anneal at a temperate in the range of 50-60 degrees c.
Primers than anneal at a higher temperate are better because they are more specific. Forwards and reverse primers need to anneal at approximatly the same temperature. Pairs of primers shouldn't anneal to one another and a primer shouldn't have self-annealing regions either.
Types of PCR
- DNA PCR : amplification from DNA
- RNA PCR : amplification from RNA
- Multiplex PCR : different primer combinations simultaneously to detect and differentiate agents
- In situ PCR : localisation of gene/transcript
- Real time PCR : uses various types of dye chemistries.
Conventional PCR has poor precision, low sensitivity, is non automated, difficult to quantify, uses carcinogenic substances and there is lots of post-PCR processing.
Real time PCR has high precisiona and sensitivity, is highly automated, very safe and there is no extra processing.
SYBR green dye attacheds to ds DNA including primer-dimers. As more ds amplicons are produced, SYBR signal will increase. When the DNA is denatured the SYBR green dye floats free. Extension phase begins as primers anneal and the dye binds to the ds products.
- Threshold value - level of detection (ct)
- Melting temperature (Tm) - 50% DNA denatured
Cycle-threshold (ct) is directly proportional to the amount of starting template. The concentration of an unknown sample can be found using a standard curve.
Tm of a specific amplification is influenced by the length of the amplicon, nucleotide sequences of the amplicon and PCR conditions.
TaqMan probe is designed with a high-energy dye termed a Reporter at the 5'end and a low energy molecule termed a Quencher at the 3'end based on FRET (flourescent resonant energy transfer) technology. When the probe is intact the Reporters dye's emission is supressed by the Quenchers dye. When the enzyme reaches the probe, the enzyme 5' exonuclease activity begins. The Reporter starts to flouresce as it is seperated from the Quencher.
Oligonucleotide: a short polymer of nucleic acids of 50 or less bases. Primers used in PCR reactions are also called oligonucleotides.
Forward genetics: start with a mutant phenotype and work towards identifying the gene whose defect is causing that mutant phenotype.
Reverse genetics: start with a gene of interest, which is cloned, and works towards a mutant phenotype.
In-vitro mutagenesis is the production of random or specific mutations in a segment of cloned DNA. The DNA is reintroduced into an organism to assess the effects of the mutagenesis (reverse genetics). There are two approaches to mutate a plasmid in vitro:
- 1. Random - helps to indentify the location of boundaries of a particular funciton of a DNA segment. It can be used when a simple genetic screen is available. It is used as a first step when little is known about the function encoded by the DNA of interest. It narrows the focus from a large gene to a smaller region.
- 2. Site-directed - places or targets a mutation exactly where its needed. It is used to define the roles of specific sequences and provides a powerful tool for protein function analysis by allowing changes is protein structure.
In-vitro mutagenesis cont.
General strategy is to mutate the plasmid DNA in-vitro. Then transform the bacterial cells with the mutated plasmid DNA. Screen for colonies that contain mutant plasmid DNA and test for function using genetic screen or selection.
Identify a restriction site in the region of interest to be mutated. Cut the plasmid DNA with the restriction enzyme present in the area. Manipulate the linear fragment using either of these strategies:
- S1 nuclease removes single stranded nucleotides to leave a blund end
- DNA polymerase + dNTPs are added to fill in the sticky ends
Ligate the blunt ends. The restriction site is eliminated.
- Linker insertion mutagenesis =
- Treat the plasmid DNA with low concentrations of DNAase1 in the presence of Mn. Under these conditions, the enzyme cuts dsDNA in a random way.
- Ligate the cut DNA to linkers containing a restriction enzyme site.
- Enzymes restrict the linear DNA with linkers attached to create sticky ends.
- Re-circularise the plasmid DNA using a ligase.
- Tranform competent cells with the plasmid generated.
In-vitro mutagenesis cont..
- Unidirectional - use an exonuclease3 enzyme to preferntially digest the 3'end of a linea DNA molecule with 5' protruding nucleotide. Cleave with enymes that produce 5'overhands. Digest with exonuclease3. Remove the remaining ** tail with S1 nuclease.
- Bidirectional - linearise the plasmid by restriction enzyme digestion at the 5' side of the gene of interest. Treat with exonuclease to digest the DNA from both sides of the molecule. Remove a sample and stop the reaction at different time points. Ligate DNA molecules with an EcoR1 liker, digest with EcoR1 to create sticky ends, and ligate into a new vector with compatible sticky ends. Transform into competent cells.
- Oligonucleotide-directed mutagenesis - 1) incorporates a mutant oligo into one strand of plasmid DNA. The oligo is flanked by 8-12nt of the wild-type sequence on either side. 2) The sequence of the oligo is complementary to the template except for the nucleotides that define mutation. 3) Both strains replicate and segregate into seperate mutant and wild-type plasmids. 4) When the plasmids are introduced into cells, the mismatch repair system often repairs the mutated base to the complimentary base in the wild-type strand before it has a chance to replicate. So the mutant plasmids are underrepresented relative to the wild-type plasmid. 5) The mutated plasmids can be enriched with methods that destroy the wild-type template strand of DNA by using a bacterial mutant strain that contains a degrading enzyme that attacks the wild-type DNA.
In-vitro mutagenesis cont...
- Isolate plasmid DNA from a bacteria strain with DNA methylase activity
- Add mutagenic oligonucleotide primers and anneal
- Extend mutagenic oligonucleotide primers
- Digest PCR products with Dpn1 which cuts only at methylated GATC sites
- Transform into bacteria, sequence DNA to verify that the mutation has been created
The plasmid DNA is restricted with two different enzymes to have the target and to remove a small wild-type sequence. Two synthetic oligos are ligated that contain the mutant sequence and compatible directed ends. Ligate the cassette with the original plasmid and transform into competent cells.
A mutation can be introduced anywhere in a PCR produced DNA fragment. Introduce a single base mismatched between an amplification primer and a template sequence. Carry out 2 PCR reactions to produce 2 overlapping DNA fragments that bare the same mutation in the overlap region. The overlap in sequence allows fragments to hybridise. One of the two hybrids is extended by DNA polymerase to produce a duplex fragment.
You can screen for colonies transformed with mutant plasmid DNA using radio labelled probes to detect bacterial colonies.