Student Study Guide Unit 3 PDF

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BIS5014-B Unit 3 – DNA analysis and how to generate recombinant DNA (rDNA) DNA analysis and generation of recombinant DNA mo  

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1973- recombinant DNA first achieved, Boyer and Cohen used bacteria and enzyme to insert foreign DNA fragment into plasmid to generate novel chimera (start of genetic engineering) Techniques of DNA isolation, DNA sequencing, vector construction, restriction enzyme cleavage of DNA, ligation, and transfection led to the cloning of individual prokaryotic and eukaryotic genes and characterising of whole genomes With chromosomes acting as libraries of genetic information 1970-80s many genes were cloned- represented a tour de force of many molecular biologists working together Changed by development of the Polymerase Chain Reaction (PCR) by Mullis in 1983 PCR- made the isolation and amplification of DNA much easier and quicker Genes could now be isolated without a cloning step by relatively inexperienced practitioners in days, rather than years, especially after DNA genomes had been sequenced This unit will be complemented by lab practical’s in which you will isolate bacterial genomic and plasmid DNA, undertake DNA gel electrophoresis and analysis, and see demonstrations of PCR and quantitative PCR (qPCR)

Table 1 Identify reasons for why genes are cloned. Reasons for cloning DNA 1. To cure diseases by gene therapy or by correcting/replacing the defective gene 2.

3. biopharmaceuticals

Explanation Replace defective proteins with functional therapeutic ones e.g. insulin, growth homrones

It allows genes and their gene products (proteins) to be easily sequenced (to see functional domains/ homology and relationships with other genes). Characterised and easily isolated/purified. Defined genetic experiments can be carried out e.g. knock out & knock in mice (transgenic mice). To add to this, it can identify diseases as DNA is our genetic blue print and ultimately many human diseases can be identified and partially characterised at DNA level. Lots of DNA is needed to sequence it and identify genes through random searches/screens. DNA cloning can be used to make human proteins with biomedical applications, such as the insulin mentioned above. Other examples of recombinant proteins include human growth hormone, which is given to patients who are unable to synthesize the hormone, and tissue plasminogen activator (tPA), which is used to treat strokes and prevent blood clots. Recombinant proteins like these are often made in bacteria.

4. gene therapy

In some genetic disorders, patients lack the functional form of a particular gene. Gene therapy attempts to provide a normal copy of the gene to the cells of a patient’s body. For example, DNA cloning was used to build plasmids containing a normal version of the gene that's nonfunctional in cystic fibrosis. When the plasmids were delivered to the lungs of cystic fibrosis patients, lung function deteriorated less quickly^22start superscript, 2, end superscript.

5. gene analysis

In basic research labs, biologists often use DNA cloning to build artificial, recombinant versions of genes that help them understand how normal genes in an organism function.

How to isolate genomic and plasmid DNA? All DNA purification techniques are based on: 1. DNA being stable and soluble in Tris-HCl buffers (pH7.5-8.5) 2. 0.1-1mM EDTA (ethylenediaminetetra-acetate) protects DNA from degradation as it chelates Mg++ which is required by nucleases (and also DNA enzymes in general) 3. DNA precipitates in 66% (v/v) ethanol in the presence of 0.3M sodium acetate, pH5.7

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This is ‘salting out’ process (ethanol precipitation of DNA) and common step to concentrate DNA or place it in a different buffer

There are three principle ty types pes of DNA: 1. Plasmid DNA Relatively small molecular weight (3-8 kb), double-stranded circular DNA that is usually super-coiled. 2. Chromosomal DNA Large molecular weight (>200 kb), double-stranded, linear DNA. 3. Viral DNA Some small (M13, 2.4kb), double- and single-stranded DNA. Some large (Lambda, 50 kb), and double-stranded. 2 different DNA isolation processes: 1. Plasmid DNA- exploits the super-coiled nature of the plasmid 2. Isolation of genomic DNA. If viral DNA is large and double-stranded, it is purified by the chromosomal DNA method If viral DNA is small and double-stranded, it is purified by the plasmid DNA method.

Complete the Table 2 for the steps in isolating genomic DNA from living cells, e.g. E. coli. See DNA cloning Chapter section 3.1-3.2.2 Table 2 Steps in Isolation of genomic DNA Step 1. Lysing cells:

Agent(s) do what? Cells to be studied are collected Lysozyme ysozyme- added to degrade cell wall Commonly used enzyme for lysing gram positive bacteria can digest cell wall and causes osmotic shock promotes cell lysis

Product Cell extract

Sodium dodecyl sulphate- an anionic detergent which disrupts cell membrane and destables all hydrophobic interactions Commonly used as a components for lysing cells during RNA/DNA extraction Also used for denaturing proteins in preparation for electrophoresis in SDSPAGE technique

Proteins broken down by adding protease RNA broken down by adding RNAase The solution is treated with saline (salt solution) to make the debris such as broken proteins, lipid and RNA to clump together The clumped cellular debris is separated from the DNA by centrifugation of the solution 2. Separation of DNA from contaminants:

Phenol or 1:1 phenol: chlor chloroform oform extra extractionction- separates mixtures of molecules based on different solubilities Phenol denatures proteins in the sample After centrifugation of the sample, denatured proteins stay in the organic phase while aqueous phase containing nucleic acid is mixed with the chloroform, that removes phenol residues from the solution

Pure DNA

Proteinase K K-- cleaves peptide bonds next to carboxylic acid group of hydrophobic amino acid residues Used to digest protein and remove contamination from preparations of nucleic acid Addition of proteinase K to nucleic acid preparations rapidly inactivates nucleases that might otherwise degrade the DNA/RNA during purification Highly suited to this application, since enzyme is active in presence of chemicals that denature proteins, such as SDS/urea/chelating agents (EDTA)/sulfhydryl reagents/trypsin inhibitors etc. Proteinase K is used for destruction of proteins in cell lysates and for release of nucleic acid- very effectively inactivates DNases/RNases

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More for genomic than plasmid DNA Ribonuclease- catalyzes degradation of RNA into smaller components Catalyzes degradation of RNA into smaller components RNase effectively cleaves phosphodiester bond between 5’-ribose of nucleotide and phosphate group attached to 3’-ribose of an adjacent pyrimidine nucleotide which forms a 2’,3’-cyclic phosphate which is then hydrolysed to the corresponding 3’-nucleoside phosphate 3. Purification

Ion exch exchange ange chromatograph chromatography y- to separate mixture into its various components so DNA is removed from proteins and RNA in the extract Chromatography process that separates ions and polar molecules based on their affinity to the ion exchanger. Works on any type of charged molecule. However must be done in conditions that are one unit away from the isoelectric point of a protein.

4. Ethanol precipitation in the presence of sodium acetate @ 0 or -20oC

Silica matrix matrix-- placed in column and cell extract is added DNA binds to the silica Guanidium HCl (GuHCl) acts as a chaotrope This allows positively charged ions to form a salt bridge between the negatively charged silica and negatively charged DNA backbone in high salt concentration Silica bound to DNA retained in the column Whereas denatured biochemicals pass straight through absolute ethanol efficiently precipitates polymeric nucleic acids with a thick solution of DNA- ethanol can be layered on top of the sample, causing molecules to precipitate at the interface

a - by spooling

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b - by centrifugation

Aqueous phase must be removed carefully- to avoid getting interface layer (done with pipette carefully) Better DNA preps are spooled o Chromosomal DNA can also be precipitates by adding 1/10th volume 0.3M sodium acetate and 3 x volumes of absolute ethanol o Spin 120000 rpm for 10 minutes o o

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Wash pellet with 70% (v/v) ethanol Air dry 15 minutes

o Resuspend DNA in 10mM Tris-HCl/0.1 mM EDTA Spooling chromosomal DNA o DNA precipitates as a stringy mass and can be spooled onto a glass rod o Spooling purifies large fragments of DNA (>50kb)

Spooled DNA appearance; in solution DNA adds viscosity (like oil, air bubbles get trapped and go backwards) These steps apply to isolation of genomic DNA from most cells, the only difference is how cells are treated to made cell extracts, e.g. guanidinium thiocyanate disrupts animal cell walls without affecting DNA binding to silica matrix spin columns Plant tissues are rich in carbohydrates and here a detergent is used CTAB (cetyltrimethylammonium bromide) to precipitate DNA away from plant extracts and the DNA is released in 1M NaCl before ethanol precipitation, see Chapter section 3.1.6. o

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Isolation of plasmid DNA Plasmids being much smaller can be isolated from cell extracts via a cleared lysate or on the basis of their conformation, see Chapter 3.2

Describe how plasmid DNA is isolated ffrom rom cleared lysates  Relies on very gentle lysis of bacterial to release small molecules  Including very compact, supercoiled plasmid into solution  While trapping larger molecules such as chromosomal DNA fragments in the remains of the cells  High speed centrifugation of pellets cell debris and trapped chromosomal DNA  To produce a cleared lysate highly enriched for plasmid Describe how plasmid DNA is isolated ffrom rom alkaline lysates (don't cov cover er CsCl gr gradients adients - a worthy technique, but now consigned to history history,, as kits allow the plasmid DNA to be rrapidly apidly purified on silica matri matrices) ces)  The basis of this technique is a narrow pH range at which non-supercoiled DNA is denatured- whereas supercoiled plasmids are not  If NaOH is added to a cell extract or cleared lysate, so that the pH is adjusted to 12-12.5 (pH rises due to NAOH)  then the hydrogen bonding in non-supercoiled DNA molecules is broken  causing the double helix to unwind and the two polynucleotide chains to separate  If the acid is now added these denatured bacterial strands reaggregate, leaving plasmid DNA in the supernatant  Under certain conditions most of the protein and RNA becomes insoluble and can be removed by the centrifugation step. Step-by-step pr procedure ocedure inv involved olved in the process: 1. Cultivate bacterial sample. 2. Resuspend the pelleted cells in buffer solution. 3. Lyse the cells. 4. Neutralize the solution with potassium acetate. 5. Clean it up. Isolate your plasmid DNA by using a process known as ethanol precipitation 5. How to analyze DNA? DNA can be analyzed in a number of ways. To determine the presence of, and to visualize the nature and integrity of the DNA the primary technique is submarine gel electrophoresis, see next diagram and read Chapter section 4.2.6. In this technique negatively charged DNA moves through a matrix/sieve of agarose in a snake-like manner (reptation ) towards the positive. The smaller molecules get through quicker that the larger molecules. DNA samples are run alongside DNA markers of known sizes (measured in base-pairs, bp, or kilobase-pairs kbp)

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The DNA molecules are visualized using stains such as Gel Red, which are non-toxic. Previously, ethidium bromide was used but this was a mild carcinogen. All of the dyes require illumination to visualize the DNA and some use light in the UV range (which can cause severe burns!). DNA fragment size in practice is determined by running your samples alongside DNA markers of known size and estimating the DNA size, or plotting a log mol. mass. in base-pairs versus distance travelled from the origin, see DNA cloning Fig 4.14. DNA runs on an agarose gel according to both size and conformation. Conformation is only an issue for super-coiled plasmids, see next figure over the page. We can measure DNA concentration against a standard concentration of DNA, but usually measure concentration by spectroscopy at OD260nm in quartz cuvettes. 1 OD260nm = 50 g/ml of pure dsDNA. DNA stains can typically identify >0.01 g of DNA, and generally 1  g of DNA is used for cloning or visualization of DNA.

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You will prepare bacterial genomic and plasmid DNA to analyze in the practicals. Component of 6X gel loading dy dye e 40% Sucrose 0.17% Xylene Cyanol 0.17% Bromophenol blue

Function Makes the sample denser than the samp sample le buffer so the sample will remain in the bottom o off the well rather than float floating ing out Makes a lower dye fr front ont Makes an upper dye ffront ront

*2 dye fronts help the user to monitor rate of migration and to prevent the over running of the gel*

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6. Enzymes used to produce recombinant DNA Read Chapter 4 of Gene cloning to see the range of enzymes used to manipulate DNA (nucleases, ligases, polymerases and modifying enzymes that add or remove chemical groups). Complete Table 3 to list the enzymes involved in manipulating DNA, briefly outline their properties and applications. See Chapter 4 of Gene cloning

Enzyme Bal31 nuclease

E. coli Exonuclease III S1 nuclease

DNase I

Type II restriction enzymes

E. coli DNA pol I. Klenow DNA pol.

Taq DNA pol.

Alkaline phosphatase

T4 polynucleotide kinase Terminal deoxynucleotidyl transferase

Ke Keyy Properties and cloning app application lication Exonuclease and endonuclease Requires calcium for an essential cofactor Degrades double stranded DNA and RNA Capable of cleaving at DNA/RNA nicks and gaps Catalyzes stepwise removal of monomic from 3’-OH termini of Ds DNA Endonuclease Preferentially targets RNA over RNA Cleaves single stranded molecules Recombinant endonuclease Stable at its optimal (neutral pH) range Suitable for RNA preparation at neutral Non-specifically degrades single stranded nucleic acids Introduction of single stranded nicks and breaks in duplex DNA, RNA and DNA/RNA Endonuclease Not require ATP Cut at specific nucleotide sequences Exonuclease Repairs any damage within DNA Large produced Proteolytic product of E. coli DNA poly 1 Exonuclease activity Random primer labelling Thermostable Adds additional nucleotide (usually adenosine) to end of each strand it synthesizes Homodimer protein enzyme 86kDa Optimally active at alkaline pH environment Removes phosphate group present at 5’ terminuses of a DNA molecule Inactivated by heat @ 68˚C for 10 minutes Adds phosphate groups onto free 5’ termini Doesn’t require a template Regulation of it occurs via multiple pathways Adds one or more deoxyribonucleotides onto 3’ terminus of a DNA molecule

NEB catalogue detail for BamH1:

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Which organism it is isolated from? Bacillus amyloliquesfacients H Which NEB buffer is optimal for digestion? BSA 150Mm NaCL, 10Mm Tris-HCl, 10Mm MgCl, 1Mm dithiothreitol supplemented with 100 microgram/ml BSA. What type of ends does the digest produce? Digest produces sticky ends Why is it necessary to supplement the reaction with BSA (bovine serum albumin)? For enzyme stability and because signals of it increase in assays. Why is 50-fold over-digestion and subsequent ligation a measure of quality? What is the potential problem? 50-fold digestions- quantitative determination of enzyme purity and lack nonspecific DNases. Ligation- determine the functional purity of the DNA after restriction enzyme digestion. Why is this enzyme available in 2 concentrations? When would you use the low concentration and when the high concentration? High concentrations of BamH 1 can cause star activity so low concentrations are used. If ionic strength is high then high concentration can be used. Incomplete digestion can occur when other too much or too little enzymes are used. What is star activity? Star activity is cutting at the wrong site (high glycerol conc, salt etc). Is the relaxation or alteration of the specificity of restriction enzyme mediated cleavage of DNA that can occur under reaction conditions that differ significantly from those optimal for the enzyme. How would you heat inactivate this enzyme? Why would you want to heat inactivate? Heat denatures the enzyme by changing its tertiary structure What is 1 unit of activity, and how much would you use in a reaction to cut 1 g of plasmid DNA with a unique BamHI site? 1 unit cuts 1 microgram of plasmid with a single site in one hour General questions on restriction enzymes Statistically how often would you expect enzyme that recognized a 4bp sequence, and an enzyme that recognized a 6bp sequence, to cut genomic DNA. Draw an example of a 3’-overhang, 5’-overhang, 3’-recess, 5’-recess and blunt end double-stranded molecule. How does a restriction enzyme identify its restriction site? How does a bacterium prevent it’s own DNA being cleaved? Construct a restriction map of the linear 15 kb DNA fragment from the data given in the table. (Two maps, mirror images of each other can be drawn, either version is acceptable) Enzymes KpnI PstI SmaI KpnI + PstI KpnI + SmaI SmaI + PstI

Restriction fragment length 5 kb, 10 kb 2 kb, 13 kb, 1 kb, 4kb, 10kb, 2kb, 3kb, 10kb 2 x 1kb, 4kb, 9kb 1kb, 2 x 2kb, 10kb

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Try this using graph paper drawing fragments to scale, or even using trimmed sections of colored post-it notes to consider all possible combinations of restriction sites. Usual only one of the combinations fit! It is a bit like doing a jigsaw puzzle! Some restriction enzymes recognize the same DNA sequence and are known as isoschizomers. See https://www.neb.com/tools-and-resources/selection-charts/isoschizomers

This can be exploited to use other enzymes to generate compatible ends, e.g. BamHI generates 5’-GATC overhangs, so any enzyme that generates 5’-GATC overhangs would be compatible to ligate together. 7. How to introduce DNA into living cells See Chapter 5 of Gene cloning, sections 5.1, 5.2, and 5.5. Cloning / making recombinant DNA is a general description of making viable novel DNA molecules, but strictly speaking the ‘clone making’ step refers to the latter experimental stages in which DNA is introduced into host cells. Bacterial cells are usually the most common host, and E. coli K-12 disabled strains (recA mutants and mutants defective at modifying DNA) are used as they survive poorly in the environment, and have in ~70 years of research never caused infection to any labworker. How do we get naked DNA into cells? What happens to it, and how can we monitor it? These are 3 important questions. DNA is negatively charged and doesn't pass through membranes, so we need to treat cells to enhance the uptake of DNA, see next activity and search on the internet. The introduction of DNA into cells is known as transformation for genomic DNA, and transfection for viral DNA, though this distinction is often blurred at times. Since few cells are naturally competent for uptake of DNA, a variety of artificial methods are used, including chemical methods, enzymes, direct injection and ballistic methods! Complete Table 4 below on the methods used to introduce DNA into cells

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Transformation Method CaCl2 treatment of E. coli

Outline how DNA gets in to cells Causes DNA to precipitate onto the outside of the cell salts responsible for change in cell wall improves DNA binding DNA becomes attached to exterior of cell and is stimulated into cell by heat shock
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