Lecture 3 - BIO 311 DNA and Restriction Digest Enzymes That...

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Unformatted text preview: BIO 311 DNA and Restriction Digest Enzymes That Cut Protease enzymes that cut proteins RNases enzymes that cleave RNA DNases enzymes that cut DNA 1. 2. Exonucleases: enzymes that cleaves off nucleotide residues at the ends of the nucleic acid strand Endonucleases: enzymes that cleaves the phosphodiester bond within the nucleic acid chain Endonucleases http://www.brownlab.info/ http://rbc.gsim3.fr/ http://gibk26.bse.kyutech.ac.jp Endonucleases protect host bacterial cell from phage infection by cutting invader's DNA Micklos et al, DNA Science, p. 109 Digest by type II endonucleases A type II endonuclease will cut the phosphate backbone on both strands.... ... at a specific palindromic sequence 5' P Phosphodieste r backbone 3' Endonuclease Restriction Site BamHI 5'GGATCC3' 3'CCTAGG5' Palindrome: "Never odd or even" S 3' 5' Type II restriction endonucleases Micklos et al, DNA Science p. 111 Exonucleases cuts: 5' overhang, 3' overhang and blunt ends Eco R1 5' overhang 5' G A A T T C 3' 3' C T T A A G 5' 5' G3' 5' A A T T C 3' 3' C T T A A 5' 3' G 5' "sticky ends" 5' G G T A C 3' 5' C 3' 3' C 5' 3' C A T G G 5' KpnI 3' overhang 5' G G T A C C 3' 3' C C A T G G 5' Sma I blunt end 5' C C C G G G 3' 3' G G G C C C 5' 5' C C C 3' 5' G G G 3' 3' G G G 5' 3' C C C 5' "Sticky Ends in English Words?" 1. Banana B anana ananaB 2. Dresser D resser resserD 3. Grammar G rammar rammarG 4. Potato P otato otatoP 5. Revive R evive eviveR 6. Uneven U neven nevenU 7. Assess A ssess ssessA Question: Which type of end does BamH1 produce? 1. 5' overhang 3' overhang Blunt end G \/ G A T C C 1. 1. Question: Which type of end does PstI produce? 1. 5' overhang 3' overhang Blunt end CTGCA \/ G 1. 1. Question: Which type of end does HaeIII produce? 1. 5' overhang 3' overhang Blunt end G G \/ C C 1. 1. Isoschizomers and Isocaudomers: Isoschizomers: endonucleases cutting the same recognition site. Example: Sma I (GGG^CCC) and Xma I (G^GGCCC) HpaII (C^CGG) and MspI (C^CGG) Isocaudomers: endonucleases cutting different recognition site, but producing same overhang extensions. Example: Bam HI (C^GATCC) and Sau3AI (^GATC) Why use endonucleases? cDNA GGATCC CCTAGG GGATCC CCTAGG BamHI digest GATCC G G CCTAG Plasmid Add BamHI digested plasmid G CCTAG GATCC G Plasmid "sticky ends" GGATCC CCTAGG "sticky ends" GGATCC CCTAGG DNA fragments cut by endonucleases can be separated using agarose gels 1+ Kb Restriction fragments can be separated and analyzed by electrophoresis on an agarose gel Scanning EM image of agarose polymer http://openwetware.org http://openwetware.org 1 Kb plus DNA Standard The DNA standards contain linear pieces of DNA of known size. Pictures of the DNA standards used in this course are found in the Appendix of the Lab Manual The size in base pairs (bp) is written next to most bands. Notice the distinct pattern of this DNA standard (Purchased from Invitrogen) 3 2 8 pET vectors? http://www.bio.davidson.edu/ "The pET System is the most powerful system yet developed for the cloning and expression of recombinant proteins in E. coli." (Novagen) Advantages of pET vectors Created for gene expression in bacteria The inserted gene is controlled by inducible T7 expression system. The codons of the inserted gene can easily be positioned inframe with vectorencoded tags (such as His tag) pET system includes variety of vectors with different antibiotic resistances and tags for affinity purification and antibody detection. Variety of pET vectors Look in pET Manual for Table pEGFP is the source of the cDNA for Enhanced Green Fluorescent Protein (EGFP) BamH1 2. 1. pUC ori pUC 19 MCS Plac selection Ampr * 1. 3. 1. 4. EcoR1 1. 2. 1. *The antibiotic resistance genes are always ON Structure of the Green Fluorescent Protein The fluorophore is in the center of the -can GFP emits bright green under "black" (UV) light (Exitation 488, Emission 509) http://www.ece.neu.edu/ GFP expressing organisms http://papilio.ab.a.utokyo.ac.jp http://www.mongabay.com/ Creation of pET28bEGFP clone through directional cloning Bam H1 cut r fo NA P cD EGF R T4 DNA ligase Eco R1 joining Eco R1 cut AMP KANR T7 promoter Bam H1 joining pEGFP vector cut with Bam H1 and Eco R1 to excise cDNA for EGFP pET28b vector cut with Bam H1 and Eco R1 accepts cDNA for EFGP *The antibiotic resistance genes are always ON Directional cloning GOI Cut the plasmid containing the gene of interest with two incompatible restriction enzymes GOI = Gene of Interest We use two incompatible restriction enzymes: EcoRI and BamHI EcoR1 5'- GAATTC CTTAAG -5' EcoR1 5'- AATTC G G CTTAA -5' BamH1 5'- GGTACC CCATGG -5' BamH1 5'- GTACC G G CCATG -5' EcoR1 5'- AATTC X BamH1 CCATG -5' EGFP cDNA will be inserted in a predetermined orientation YES NO We used restriction endonucleases to digest DNA....what's the next step? Next steps in cloning a gene: Lab B 4 Ligation of fragments into an expression plasmid Lab B5 Transformation of this DNA into bacteria Cloning and amplification of cDNAs Cut with Restriction Enzyme Cloning Vector (pET28) cDNA (EGFP) Recombinant Vector (DNA to be cloned) Ligation Linear vector Digested cDNA (DNA Ligase) Transformation & Amplification in Bacteria Restriction Digestion or PCR Plasmids and bacterial chromosome How many genes are on the plasmid DNA? How many genes are on the Bacterial DNA? http://citnews.unl.edu/ Genome Comparisons organism Homo sapiens (human) Rattus norvegicus (rat) Mus musculus (mouse) Drosophila melanogaster (fruit fly) Arabidopsis thaliana (plant) Caenorhabditis elegans (roundworm) Saccharomyces cerevisiae (yeast) Escherichia coli (bacteria) H. influenzae (bacteria) estimated size 2900 million bases 2,750 million bases 2500 million bases 180 million bases estimated gene number ~30,000 ~30,000 ~30,000 13,600 # of Chromosomes 46 42 40 8 125 million bases 97 million bases 25,500 19,100 10 12 12 million bases 6300 32 4.7 million bases 1.8 million bases 3200 1700 1 1 http://www.ornl.gov/sci/techresources/Human_Genome/faq/compgen.shtml#genomesize I Summary of the specific cloning steps 1. V 1. 2. 3. 4. 5. *The antibiotic resistance genes are always ON Use restriction enzymes (EcoRI and BamHI) to cut a DNA fragment with the GOI (EGFP) (insert = I) Cut a receiving vector (pET28, V) with the same restriction enzymes Join the insert to the vector using DNA ligase to make pET28bEGFP Transform bacteria with the recombinant plasmid Select the transformed bacteria on agar with appropriate antibiotics* Analyze colonies for desired recombinant plasmid pET28b-EGFP ...
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