Restriction Enzyme Digestion & Southern Blotting of DNA
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1 Restriction Enzyme Digestion & Southern Blotting of DNA
2 Experiment Goals Digestion of DNA by restriction enzyme Analyze digested DNA by electrophoresisTransfer digested DNA to nitrocellulose filters (Southern blotting)Procedure of setting up a Southern blotting
3 Restriction Enzymes Definition: A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA at specific sites that it recognizes.The enzyme makes two incisions, one through each of the sugar-phosphate backbones (i.e., each strand) of the double helix without damaging the nitrogenous bases.Often the recognizes sites are palindromic.N is the length of the genome and b is the restriction site length.
4 Restriction enzymeThe enzyme EcoRI cutting DNA at its recognition sequenceDifferent restriction enzymes have different recognition sequences.This makes it possible to create a wide variety of different gene fragments.
6 Function of Restriction Enzymes in microorganisms Provide microorganisms with resistance to invading organisms or foreign DNA.Endonucleases in bacterial cells resist infections by viruses, by destroying foreign DNA molecules.consist of a related pair of enzymesEndonuclease – cuts foreign DNAMethylase – protects host DNA
7 Methylase EnzymesRestriction enzymes usually occur in combination with one or two modification enzymes (DNA-methyltransferases)Protect the cell’s own DNA from cleavage by the restriction enzyme.Modification enzymes recognize the same DNA sequence as the restriction enzyme that they accompany,Instead of cleaving the sequence, they methylate one of the bases in each of the DNA strands.The methyl groups protrude into the major groove of DNA at the binding site and prevent the restriction enzyme from acting upon it.
8 NamingRestriction enzymes are named based on the bacteria in which they are isolated in the following manner: example “EcoRI”E Escherichia (genus)co coli (species)R RY13(strain)I First identified Order
9 Restriction EnzymeThere are hundreds of different REs from different microorganismsEach RE cuts DNA at a specific “recognition sequence” of nucleotides. Examples:EcoRI-- GAATTC;AluI -- AGCTEach recognizes its specific “recognition sequence” and cuts both strands of DNA wherever that sequence is found, but nowhere else.
10 Restriction Enzyme Uses Recombinant DNA technologyCloningReplicates a sequence inserted into a host cellDNA restriction mappingA rough map of a DNA fragmentDNA fingerprints
11 Types of Restriction Enzymes Restriction enzymes are traditionally classified into three types on the basis ofsubunit composition,cleavage position,sequence-specificityand cofactor-requirements
12 Types of Restriction Enzymes Type I - Recognize specific sequences and cut DNA at a nonspecific site > than 1,000 bp awayType II - Recognize palindromic sequences and cut within the palindromeType III - Recognize specific 5-7 bp sequences and cut bp down stream of the site.Type II restriction enzymes are the most useful class as they recognize specific palindomic sequences in DNA and cut the sugar phosphate backbone within the palindrome
13 Restriction Enzymes and DNA fragments A restriction enzyme functions by "scanning" the length of a DNA molecule.Once it encounters its particular specific recognition sequence,it will bind to the DNA moleculeand makes one cut in each of the two sugar-phosphate backbones of the double helix.
14 Endonucleases and DNA fragments Blunt endsSticky ends
15 Unit Determination Assay One unit of restriction endonuclease is defined as the amount of enzyme required to digest one microgram of the appropriate substrate DNA completely in 60 minutes under the conditions specified for that enzyme.
16 Set up of a restriction enzyme reaction A RE reaction contains the DNA to be analyzed,A restriction enzyme,A restriction enzyme buffer mix.contains a buffering agent to maintain constant pH,and Mg++ (from MgCl2) as a necessary cofactor for enzyme activity.
20 Southern BlottingThe technique was developed by E.M. Southern in 1975.
21 What Is Southern Blotting? A technique used in molecular biology to check for the presence of a particular DNA sequence in a DNA sample.
22 Southern BlotThe Southern Blot takes advantage of the fact that DNA fragments will stick to a nylon or nitrocellulose membrane.The membrane is laid on top of the agarose gel and absorbent material (e.g. paper towels or a sponge) is placed on top.With time, the DNA fragments will travel from the gel to the membrane by capillary action as surrounding liquid is drawn up to the absorbent material on top.The membrane is now a mirror image of the agarose gel.
23 Uses of Southern Blotting Identify mutations, deletions, and gene rearrangementsUsed in prognosis of cancer and in prenatal diagnosis of genetic diseasesdiagnosis of Leukemiasdetect variations in gene structureidentify homologous genes among different species
24 Performing Southern Blotting DNA Digestion with an appropriate restriction enzyme.Gel Electrophoresis run the digest on an agarose gel.Denature the DNA (usually while it is still on the gel).Transfer the denatured DNA to the membrane (blotting)Preparing the probeHybridization Probe the membrane with labeled ssDNA.Detection Visualize your radioactively labeled target sequence.
25 1- DNA Digestion Cut the DNA into different sized pieces. HinfI restriction enzyme is used
26 2- Gel Electrophoresis Sorts the DNA pieces by size Agarose or polyacrimide
27 Electrophoresis of Genomic DNA Odd numbered lanes contain undigested genomic DNAEven numbered lanes contain digested genomic DNA
28 3- Denature the DNADNA is then denatured with an alkaline solution such as NAOH.This causes the double stranded to become single-stranded.
29 4- Blotting Transfer the DNA from the gel to a solid support. The blot is usually done on a sheet of nitrocellulose paper or nylon.Transferred by either electrophoresis or capillary blotting.
30 4- Blotting1) Electrophoresis- takes advantage of the molecules negative charge.
32 4- Blotting2) Capillary blotting-fragments are eluted from the gel and deposited onto the membrane by buffer that is drawn through the gel by capillary action.BufferWick (filter paper)Filter paperAgar gel with DNAMembraneWeightPaper towel stack
33 4- Blot Fixation The blot is made permanent by: Drying at ~80°C Exposing to UV irradiation
34 5- Preparing the probeIt is a fragment of DNA of variable length (usually bases long), which is used to detect in DNA the presence of nucleotide sequences that are complementary to the sequence in the probeMust be labeled to be visualizedUsually prepared by making a radioactive copy of a DNA fragment. Probing is often done with 32P labeled ATP, biotin/streptavidin or a bioluminescent probe.
35 6- HybridizationHybridization-process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target patient DNA.
36 6- Hybridization Steps for hybridization 1. The labeled probe is added to the matrix incubated for several hours to allow the probe molecules to find their targets2. Any unbound probes are then removed.3. The place where the probe is connected corresponds to the location of the immobilized target molecule.
38 7- Detection Visualize your labeled target sequence. If radiolabeled 32P probe is used, then you would visualize by autoradiography.Biotin/streptavidin detection is done by colorimetric methods,and bioluminescent visualization uses luminescence.
39 Steps in Southern Blotting DNA extractionDisease geneFragments ofDNA appearas a smearDNA digestionGel electrophoresisPaper towelsThis process takes ~8 daysChromatographypaper supportDenaturation of patient’s DNA in gelGel inNaOHNylon filterSouthern blotGel10x SSCBlot dismantledAutoradiographyX ray filmHybridisation:Stringency washesRadioactive probeadded to filtercassettefilterfilter
40 Southern Blot of same DNA (final result on x-ray film)
42 Advantages and Disadvantages of Southern Blotting The exact location of a disorder does not need to be knownDisadvantagesThey are rather time consumingNeeds high quality/quantity DNA RadioactiveSome tasks previously carried out by Southern blot are now done using the polymerase chain reaction (PCR) which is faster