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!!!