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Restriction Digestion and Restriction Digestion and Analysis of Lambda DNA KitAnalysis of Lambda DNA Kit
What can you do with the Restriction What can you do with the Restriction Digestion and Analysis of Lambda DNA Digestion and Analysis of Lambda DNA
Kit?Kit?
Understand the use of restriction enzymes as Understand the use of restriction enzymes as biotechnology toolsbiotechnology tools
Become familiar with principals and techniques Become familiar with principals and techniques of agarose gel electrophoresisof agarose gel electrophoresis
Generate a standard curve from a series of DNA Generate a standard curve from a series of DNA size fragmentssize fragments
Estimate DNA fragment sizes from agarose gel Estimate DNA fragment sizes from agarose gel datadata
What are restriction enzymes?What are restriction enzymes?
Evolved by bacteria to protect against viral Evolved by bacteria to protect against viral DNA infectionDNA infection
Endonucleases = cleave within DNA strandsEndonucleases = cleave within DNA strands
3,139 known enzymes3,139 known enzymes
How does it work?How does it work?Enzyme Site RecognitionEnzyme Site Recognition– Each enzyme digests (cuts) Each enzyme digests (cuts)
DNA at a specific sequence DNA at a specific sequence restriction siterestriction site
– Enzymes recognize Enzymes recognize 44-, -, 66- or - or 8-8- base pair, palindromic base pair, palindromic sequencessequences
– IsoschizomersIsoschizomers recognize recognize identical sequences, but have identical sequences, but have different optimum reaction different optimum reaction conditions and stabilitiesconditions and stabilities
– Can be Can be unambiguousunambiguous or or ambiguousambiguous
Unambiguous
Ambiguous
Palindromic SequencesPalindromic Sequences
5’ versus 3’ overhang: 5’ versus 3’ overhang: Sticky EndsSticky Ends
Enzyme cuts Enzyme cuts 5’ GAATTC 3’
3’ G 5’
5’ G 3’
3’ CTTAAG 5’
5’ G 3’
3’ CTTAA 5’
5’ AATTC 3’
3’ G 5’
Generates 5’ overhang
5’ and 3’ versus Blunt ends
Common Restriction EnzymesCommon Restriction Enzymes
EcoEcoRIRI– EscherichiaEscherichia colicoli– 5’ overhang5’ overhang
HindIIIHindIII– Haemophilus influensaeHaemophilus influensae
– 5’ overhang5’ overhang
PstPstII– ProvidenciaProvidencia stuartiistuartii– 3’ overhang3’ overhang
5’ CTGCAG 3’
3’ CACGTC 5’
5’ GAATTC 3’
3’ CTTAAG 5’
5’ AAGCTT 3’
3’ TTCGAA 5’
What is needed for restriction digestion?What is needed for restriction digestion?
Template DNATemplate DNA, uncut DNA, often bacterial , uncut DNA, often bacterial phage DNAphage DNA
DNA standard or markerDNA standard or marker, a restriction , a restriction enzyme of known fragment sizes enzyme of known fragment sizes
Restriction enzyme(s)Restriction enzyme(s), to cut template , to cut template DNA DNA
Restriction BufferRestriction Buffer, to provide optimal , to provide optimal conditions for digestionconditions for digestion
Lambda Phage DNALambda Phage DNA
Genomic DNA of a bacterial virus
Attacks bacteria by inserting its nucleic acid into the host bacterial cell
Replicates rapidly inside host cells until the cells burst and release more phages
Harmless to man and other eukaryotic organisms
Restriction Enzyme DigestionRestriction Enzyme Digestion
Restriction BufferRestriction Buffer provides optimal provides optimal conditionsconditions
– NaCl NaCl provides the correct ionic strengthprovides the correct ionic strength
– Tris-HClTris-HCl provides the proper pH provides the proper pH
– MgMg2+2+ is an enzyme co-factor is an enzyme co-factor
Why incubate at 37Why incubate at 37C?C?Body temperature is optimal for these and Body temperature is optimal for these and mostmost
other enzymesother enzymes
What happens if temperature is too hot What happens if temperature is too hot or cool? or cool? Too hot = enzyme may be denatured, killedToo hot = enzyme may be denatured, killed Too cool= enzyme activity lowered, requiring Too cool= enzyme activity lowered, requiring
longer digestion timelonger digestion time
DNA Digestion Temperature DNA Digestion Temperature
Agarose Gel ElectrophoresisAgarose Gel Electrophoresis
Electrolysis:Electrolysis: the splitting of water using the splitting of water using electricityelectricity– current splits water into hydrogen ions (Hcurrent splits water into hydrogen ions (H++) )
and hydroxyl ions (OHand hydroxyl ions (OH--))
ElectrophoresisElectrophoresis:: a method of separating a method of separating charged molecules in an electrical field; charged molecules in an electrical field; DNA has an overall negative chargeDNA has an overall negative charge
Used to separate DNA fragments by sizeUsed to separate DNA fragments by size
Components of an Electrophoresis SystemComponents of an Electrophoresis System
Power supply and chamberPower supply and chamber, a source of negatively , a source of negatively charged particles with a cathode and anodecharged particles with a cathode and anode
BufferBuffer,, a fluid mixture of water and ions a fluid mixture of water and ions
Agarose gelAgarose gel, a porous material that DNA migrates , a porous material that DNA migrates throughthrough
Gel casting materialsGel casting materials
DNA ladderDNA ladder, mixture of DNA fragments of known , mixture of DNA fragments of known lengthslengths
Loading dyeLoading dye, contains a dense material and allows , contains a dense material and allows visualization of DNA migrationvisualization of DNA migration
DNA StainDNA Stain, allows visualizations of DNA fragments , allows visualizations of DNA fragments after electrophoresisafter electrophoresis
Buffer
Dyes
Power Supply
+
-
Agarosegel
Cathode
Anode
Bio-Rad’s Electrophoresis Equipment
Precast Ready Agarose Gel
Power Supplies
Electrophoresis BufferElectrophoresis Buffer
TAE (Tris-acetate-EDTA) and TBE (Tris-TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA) are the most common borate-EDTA) are the most common buffers for duplex DNAbuffers for duplex DNA
Establish pH and provide ions to support Establish pH and provide ions to support conductivityconductivity
Concentration affects DNA migrationConcentration affects DNA migration– Use of water will produce no migratonUse of water will produce no migraton– High buffer conc. could melt the agarose gelHigh buffer conc. could melt the agarose gel
Agarose GelAgarose Gel
A porous material derived from red A porous material derived from red seaweedseaweed
Acts as a sieve for separating DNA Acts as a sieve for separating DNA fragments; smaller fragments travel fragments; smaller fragments travel faster than large fragmentsfaster than large fragments
Concentration affects DNA migrationConcentration affects DNA migration
– Low conc. = larger poresLow conc. = larger pores better better resolution of larger DNA fragmentsresolution of larger DNA fragments
– High conc. = smaller poresHigh conc. = smaller pores better better resolution of smaller DNA fragmentsresolution of smaller DNA fragments
DNA StainingDNA Staining
Allows DNA visualization after gel Allows DNA visualization after gel electrophoresiselectrophoresis
Ethidium BromideEthidium Bromide
Bio-Safe DNA stainsBio-Safe DNA stains
Agarose Gel
DNA Fragments
Complete a Gel Electrophoresis simulation at:
http://gslc.genetics.utah.edu/units/biotech/gel/
Restriction Restriction Enzyme Digest Enzyme Digest and Analysis and Analysis ProceduresProcedures
Actual Results of Restriction Actual Results of Restriction Enzyme DigestionEnzyme Digestion
Lane 1, DNA markers Lane 1, DNA markers ((HinHindIII lambda dIII lambda digest)digest)lane 2, uncut lambda lane 2, uncut lambda DNADNAlane 3, lambda DNA lane 3, lambda DNA digested with digested with PstPstIIlane 4, lambda DNA lane 4, lambda DNA digested with digested with EcoEcoRIRIlane 5, lambda DNA lane 5, lambda DNA digested with digested with HinHindIIIdIII
Analysis of DNA FragmentsAnalysis of DNA Fragments
Determine restriction fragment sizesDetermine restriction fragment sizes
– Create standard curve using DNA markerCreate standard curve using DNA marker
– Measure distance traveled by restriction fragmentsMeasure distance traveled by restriction fragments
– Determine size of DNA fragments Determine size of DNA fragments
DNA Marker Standard CurveDNA Marker Standard Curve
Size (bp)Size (bp) Distance Distance (mm)(mm)
23,00023,000 11.0 11.0
9,4009,400 13.0 13.0
6,5006,500 15.0 15.0
4,4004,400 18.0 18.0
2,3002,300 23.0 23.0
2,0002,000 24.0 24.0
Factors Affecting Factors Affecting Restriction Enzyme DigestionRestriction Enzyme Digestion
Temperature, restriction enzymes are Temperature, restriction enzymes are sensitive to prolonged periods of exposure to sensitive to prolonged periods of exposure to heatheat
Cross contamination of restriction enzymesCross contamination of restriction enzymes
Buffer, optimum pHBuffer, optimum pH
Incubation temperature, maintain optimum Incubation temperature, maintain optimum temperature during restriction enzyme activitytemperature during restriction enzyme activity
And Finally…Don’t forget to ADD your And Finally…Don’t forget to ADD your restriction enzyme to the reaction!!!restriction enzyme to the reaction!!!
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