Restriction Digestion and Analysis of Lambda DNA Kit

1. Restriction Digestion and Analysis of Lambda DNA Kit

2. 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

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

4. 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

5. 5’G 3’ 3’CTTAAG5’ 5’G 3’ 3’CTTAA 5’ 5’GAATTC3’ 3’G5’ 5’ AATTC3’ 3’G5’ Palindromic Sequences • 5’ versus 3’ overhang: Sticky Ends Enzyme cuts  Generates 5’ overhang 5’ and 3’ versus Blunt ends

6. 5’GAATTC3’ 3’CTTAAG5’ 5’AAGCTT3’ 3’TTCGAA5’ 5’CTGCAG3’ 3’CACGTC5’ Common Restriction Enzymes • EcoRI • Escherichiacoli • 5’ overhang • HindIII • Haemophilus influensae • 5’ overhang • PstI • Providenciastuartii • 3’ overhang

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. Cathode - Anode + Buffer Dyes Agarose gel Power Supply

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

15. 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

16. 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

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

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

19. Restriction Enzyme Digest and Analysis Procedures

20. 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

21. 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

22. 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

23. 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!!!