Restriction Digestion and Analysis of Lambda DNA Kit

What can you do with the Restriction Digestion and Analysis of Lambda DNA Kit? Understand the use of restriction enzymes as biotechnology tools Become familiar with principals and techniques of agarose gel electrophoresis Generate a standard curve from a series of DNA size fragments Estimate DNA fragment sizes from agarose gel data

What are restriction enzymes? Evolved by bacteria to protect against viral DNA infection Endonucleases = cleave within DNA strands 3,139 known enzymes

How does it work? Enzyme Site Recognition Each enzyme digests (cuts) DNA at a specific sequence  restriction site Enzymes recognize 4-, 6- or 8- base pair, palindromic sequences Isoschizomers recognize identical sequences, but have different optimum reaction conditions and stabilities Can be unambiguous or ambiguous Unambiguous Ambiguous

Palindromic Sequences 5’ versus 3’ overhang: Sticky Ends 5’ GAATTC 3’ 3’ G 5’ 5’ G 3’ 3’ CTTAAG 5’ Enzyme cuts  5’ G 3’ 3’ CTTAA 5’ 5’ AATTC 3’ 3’ G 5’ Generates 5’ overhang 5’ and 3’ versus Blunt ends

Common Restriction Enzymes 5’ GAATTC 3’ 3’ CTTAAG 5’ EcoRI Escherichia coli 5’ overhang HindIII Haemophilus influensae PstI Providencia stuartii 3’ overhang 5’ AAGCTT 3’ 3’ TTCGAA 5’ 5’ CTGCAG 3’ 3’ CACGTC 5’

What is needed for restriction digestion? Template DNA, uncut DNA, often bacterial phage DNA DNA standard or marker, a restriction enzyme of known fragment sizes Restriction enzyme(s), to cut template DNA Restriction Buffer, to provide optimal conditions for digestion

Lambda 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 Digestion Restriction Buffer provides optimal conditions NaCl provides the correct ionic strength Tris-HCl provides the proper pH Mg2+ is an enzyme co-factor

DNA Digestion Temperature Why incubate at 37C? Body temperature is optimal for these and most other enzymes What happens if temperature is too hot or cool? Too hot = enzyme may be denatured, killed Too cool= enzyme activity lowered, requiring longer digestion time

Agarose Gel Electrophoresis Electrolysis: the splitting of water using electricity current splits water into hydrogen ions (H+) and hydroxyl ions (OH-) Electrophoresis: a method of separating charged molecules in an electrical field; DNA has an overall negative charge Used to separate DNA fragments by size

Components of an Electrophoresis System Power supply and chamber, a source of negatively charged particles with a cathode and anode Buffer, a fluid mixture of water and ions Agarose gel, a porous material that DNA migrates through Gel casting materials DNA ladder, mixture of DNA fragments of known lengths Loading dye, contains a dense material and allows visualization of DNA migration DNA Stain, allows visualizations of DNA fragments after electrophoresis Ions: atoms that have a positive or negative charge because they have lost or gained electrons. Electrophoresis: migration of ions at different speeds is a basic principal

- + Cathode Anode Buffer Dyes Agarose gel Power Supply The electrode at which electrons enter the gel box from the power supply (along the black wire) is called the cathode and is negative (-). The electrode at which electrons leave the box and re-enter the power supply (along the red wire) is called the anode and carries a positive charge (+). The flow of electrons sets up a potential energy difference between the electrodes. This is known as potential, and is measured in volts. It establishes an electric field through which the ions in the gel box fluid migrate. The migration of ions in the fluid creates electrical current which is measured in milliamperes (milliamps).

Bio-Rad’s Electrophoresis Equipment Power Supplies Precast Ready Agarose Gel

Electrophoresis Buffer TAE (Tris-acetate-EDTA) and TBE (Tris- borate-EDTA) are the most common buffers for duplex DNA Establish pH and provide ions to support conductivity Concentration affects DNA migration Use of water will produce no migraton High buffer conc. could melt the agarose gel

Agarose Gel A porous material derived from red seaweed Acts as a sieve for separating DNA fragments; smaller fragments travel faster than large fragments Concentration affects DNA migration Low conc. = larger pores better resolution of larger DNA fragments High conc. = smaller pores better resolution of smaller DNA fragments

DNA Staining Allows DNA visualization after gel electrophoresis Ethidium Bromide Bio-Safe DNA stains

Complete a Gel Electrophoresis simulation at: Agarose Gel DNA Fragments Complete a Gel Electrophoresis simulation at: http://gslc.genetics.utah.edu/units/biotech/gel/

Restriction Enzyme Digest and Analysis Procedures

Actual Results of Restriction Enzyme Digestion Lane 1, DNA markers (HindIII lambda digest) lane 2, uncut lambda DNA lane 3, lambda DNA digested with PstI lane 4, lambda DNA digested with EcoRI lane 5, lambda DNA digested with HindIII

Analysis of DNA Fragments Determine restriction fragment sizes Create standard curve using DNA marker Measure distance traveled by restriction fragments Determine size of DNA fragments

DNA Marker Standard Curve Size (bp) Distance (mm) 23,000 11.0 9,400 13.0 6,500 15.0 4,400 18.0 2,300 23.0 2,000 24.0

Factors Affecting Restriction Enzyme Digestion Temperature, restriction enzymes are sensitive to prolonged periods of exposure to heat Cross contamination of restriction enzymes Buffer, optimum pH Incubation temperature, maintain optimum temperature during restriction enzyme activity And Finally…Don’t forget to ADD your restriction enzyme to the reaction!!!