Different methods of gene isolation

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This Seminar discuss about methods of gene isolation & techniques.

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different methods of gene isolation:

S Revathi different methods of gene isolation

What are genes?:

What are genes? The basic unit of a cell that passes on the traits of parents to their children Genes are pieces of DNA. They have information for making specific proteins that control specific traits or activities . Examples of traits controlled by genes are eye color , foot size, and height . Examples of activity include the growth and repair of cells Genes are also called as alleles(traits).

Where are genes located? :

Where are genes located? Genes are actually a small string of bases on a dna chain. So a DNA chain has many genes , and these DNA are located in the chromosomes. Chromosomes are in the nucleus of all cells . In other words, Genes are part of a DNA molecule which codes for 1 polypeptide

GENE ISOLATION BASED ON PROTEIN SEQUENCE:

GENE ISOLATION BASED ON PROTEIN SEQUENCE One of the more elaborate examples of "inverse genetics" is the situation where the protein of interest has been purified, but the gene that encodes that protein has not been identified. In the first step, a portion of the protein is sequenced until a sequence of at least six contiguous amino acids is found for which the coding sequence is reasonably non-redundant . All possible coding sequences of this region are then synthesized (a nucleic acid sequence of at least seventeen nucleotides is desired for specificity reasons) in a single batch. protein sequencing by maldi-tof protein sequencing by MS

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Alternatively , one either looks at codon use by the organism and generates a "best guess" oligo, or puts inosine at positions of uncertainty. This pool of sequences is labelled and used in a Southern analysis of the entire cell's restriction enzyme-treated chromosome. Typically , one or a small number of restriction fragments will show hybridization indicating similarity to one particular probe in the pool. Having shown that there is a hybridizing sequence in the chromosome, the probe pool is then used to screen a random library of cloned chromosomal fragments nucleoside Southern blot-DNA

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Alternatively, a new library can be generated using the chromosomal fragments of the same size as the hybridizing band; this "enriched" library would then be screened with the probe pool. Any clone that shows homology to the probe would likely carry at least a portion of the gene of interest .

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At this point, there are several possibilities: ( i) the region may be sequenced in order to determine the open reading frames. Since we already know one end of the protein sequence , this will allow the conclusive identification of the desired gene . ORF: A section of a sequenced piece of DNA that begins with an initiation ( methionine ATG) codon and ends with a nonsense codon. ORFs all have the potential to encode a protein or polypeptide This sequence information can be used to clone a selectable marker (for example drug-resistance) into the coding region, for use in mutationally altering the chromosomal version.

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( ii) Alternatively, a restriction map of the cloned region can be produced that will indicate the relative position of the probe-hybridizing region within the clone . The clone can now be mutagenized with transposons and such mutations analyzed physically to choose a pair flanking the probe-hybridizing region. One of these must therefore be in the gene of interest . Before serious conclusions are drawn, a mutation in the target region should be introduced into the chromosome in order to confirm that the region really does encode the product of interest

Isolation of Nucleic Acids:

Isolation of Nucleic Acids Goals : removal of proteins DNA vs RNA isolation of a specific type of DNA (or RNA) General Features : denaturing cell lysis (SDS, alkali, boiling)  enzyme treatments protease RNase ( DNase -free) DNase ( RNase -free)

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Types of DNA : genomic (chromosomal) organellar (satellite) plasmid (extra-chromosomal) phage/viral (ds or ss ) complementary (mRNA) Types of Methods : differential solubility ‘adsorption’ methods density gradient centrifugation

High MW Genomic DNA Isolation:

High MW Genomic DNA Isolation Typical Procedure Cell Lysis 0.5% SDS + proteinase K (55 o several hours) Phenol Extraction gentle rocking several hours Ethanol Precipitation RNAse followed by proteinase K Repeat phenol extraction and EtOH

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Phenol Extraction mix sample with equal volume of sat. phenol soln retain aqueous phase optional chloroform/ isoamyl alcohol extraction(s) EtOH Precipitation 2-2.5 volumes EtOH , -20 o high salt, pH 5-5.5 centrifuge or ‘spool’ out  aqueous phase (nucleic acids)  phenol phase (proteins)

Isolation of RNA Special Considerations:

Isolation of RNA Special Considerations RNAse inhibitors extraction in guanidine salts phenol extractions at pH 5-6 ( pH 8 for DNA ) treatment with RNase -free Dnase selective precipitation of high MW forms ( rRNA , mRNA) with LiCl

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oligo-dT column- Oligo dT isolation is a very useful method for isolating sequenceswith a poly A tag http://molbio.mgh.harvard.edu/szostakweb/protocols/oligo-dt/index.html

Adsorption Methods:

Adsorption Methods nucleic acids selectively absorb to silica or resins in the presence of certain chaotropic agents or salts A chaotropic agent is a substance which disrupts the structure of, and denatures, macromolecules such as proteins and nucleic acids (e.g. DNA and RNA ). applications: plasmid preps fragments after electrophoresis PCR templates Plasmid Mini prep Protocol 1. Solubilize bacteria in alkali solution 2. Neutralize with Na-acetate 3. Centrifuge, discard pellet 4. Mix supernatant with resin + chaotropic agent 5. Wash resin 6. Elute DNA with low salt buffer

Density Gradient Centrifugation:

Density Gradient Centrifugation A procedure for separating particles such as viruses or ribosomes or molecules such as DNA in which the sample is placed on a preformed gradient such as sucrose or cesium chloride . Upon centrifugation either by rate zonal or equilibrium procedures, the macromolecules are 'banded' in the gradient and can be collected as a pure fraction. rate zonal/sucrose (size fractionation) electrophoresis more common

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20 40 60 80 % GC base pairs 1.68 1.70 1.72 1.74 density (g/cm 3 )

Isopycnic/CsCl (density) :

Isopycnic/ CsCl (density) In this type of separation, a particle of a particular density will sink during centrifugation until a position is reached where the density of the surrounding solution is exactly the same as the density of the particle. DNA ~1.7 g/cm 3 protein ~1.3 g/cm 3 RNA > DNA ssDNA > dsDNA GC content

Criteria for successful isopycnic separation: :

Criteria for successful isopycnic separation: Density of the sample particle must fall within the limits of the gradient densities . Any gradient length is acceptable . The run time must be sufficient for the particles to band at their isopycnic point. Excessive run times have no adverse effect.

CsCl Gradients:

CsCl Gradients Applications large scale preparations high purity ‘satellite’ DNA RNA ‘cushions’

Using Spectroscopy to analyze DNA:

Using Spectroscopy to analyze DNA DNA absorbs UV light with a major peak at 260 nm This absorption is useful because it varies with the structure of DNA (&RNA) i.e. extinction coefficient depends on the structure Optical Density Wave Length 260 dsDNA Low extinction coefficient ssDNA Higher extinction coefficient

Evaluation of Nucleic Acids:

Evaluation of Nucleic Acids spectrophotometrically quantity quality fluorescent dyes gel electrophoresis

Agarose Gel Stained with ethidium bromide (EtBR) to Visualize the DNA:

Agarose Gel Stained with ethidium bromide ( EtBR ) to Visualize the DNA 500 bp 600 bp 700 bp 1000 bp slots where DNA is loaded molecular weight markers molecular weight markers correct PCR product Screening PCR products to test for the presence of specific DNA sequences

Intercalating Agents Distort the Double Helix:

Intercalating Agents Distort the Double Helix Several hydrophobic molecules containing flat aromatic and fused heterocyclic rings can insert between the stacked base pairs of DNA. These molecules are called intercalating agents. Intercalating agents are potential Cancer-inducing reagents .

DNA Sequence -Dideoxy Chain Termination:

DNA Sequence -Dideoxy Chain Termination

DNA sequencing: the Sanger (dideoxy) method:

DNA sequencing: the Sanger ( dideoxy ) method

NTP, dNTPs and ddNTPs:

NTP, dNTPs and ddNTPs

DNA sequencing: the Sanger method:

DNA sequencing: the Sanger method Four separate polymerization reactions are performed

Reading a DNA Sequencing Gel:

Reading a DNA Sequencing Gel C G G G C G T Sequence 5’ to 3’

Semi-Automated Sequencing:

Semi-Automated Sequencing thermal cycler fluorescent ddNTPs unique spectra measure intensity of DNA products on gel

Automated DNA Sequencing with Fluorescent Dyes:

Automated DNA Sequencing with Fluorescent Dyes Each different ddNTP is coupled to a different colored fluorescent dye ddTTP is red; ddGTP is black etc.

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