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Genome Sizes are Large human = 3 x 10 9 bp E. coli = 4 x 10 6 bp

Modifying DNA. study of individual genes requires manipulation of nucleic acids enzymes are used to modify nucleic acids, eg.: nucleases : break down nucleic acids into smaller fragments or nucleotides polymerases : synthesize DNA (ie, copy templates)

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Genome Sizes are Large human = 3 x 10 9 bp E. coli = 4 x 10 6 bp

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  1. Modifying DNA • study of individual genes requires manipulation of nucleic acids • enzymes are used to modify nucleic acids, eg.: • nucleases: break down nucleic acids into smaller fragments or nucleotides • polymerases: synthesize DNA (ie, copy templates) • ligases: covalently join fragments (end-to-end) • Genome Sizes are Large • human = 3 x 109 bp • E. coli = 4 x 106 bp • If 1 bp = 1 mm, then: • human genome = 3000 km (1800 miles) • E. coli genome = 4 km (2.5 miles) • gene of 50 kDa protein = 2 meters

  2. Nucleases • exonucleases • remove single nucleotides from 3'- or 5'-end depending on specificity • most exhibit specificity for either RNA, ssDNA or dsDNA • good for removing undesired nucleic acid or removing single stranded overhangs from dsDNA • endonucleases • cleaves phoshodiester bonds within fragments • lack of site specificity limits uses and reproducibility

  3. EcoRI methylase Restriction Enzymes • site-specific endonucleases of prokaryotes • function to protect bacteria from phage (virus) infection • corresponding site-specific modifying enzyme (eg., methylase) • type II enzymes are powerful tools in molecular biology

  4. Features of Restriction Sites • typically 4-8 bp recognized • most are palindromes (dyad symmetry) • degeneracy permitted by some enzymes • cleavage produces 5’-PO4 and 3’-OH • both strands cleaved between same residues: • blunt ends • 5’-overhangs • 3’-overhangs

  5. Blunt End (Sma I)  -CCCGGG- -CCC GGG- ||||||  ||| + ||| -GGGCCC- -GGG CCC-  5' Overhang (Xma I)  -CCCGGG- -C CCGGG- ||||||  | + | -GGGCCC- -GGGCC C-  3' Overhang (Pst I)  -CTGCAG- -CTGCA G- ||||||  | + | -GACGTC- -G ACGTC-  • Isoschizomers • Sma I CCCGGG • Xma I CCCGGG • Compatible Ends • Pst I CTGCAG • Nsi I ATGCAT

  6. mix DNA with enzyme • DNA purity affects efficiency (RNA, proteins, salts, solvents, etc) • each enzyme has optimal conditions (eg, pH, ions, temp, etc) • double digests • enzyme order • re-purify • star activity • loss of specificity • eg, 5/6 bases Practical Considerations • Conditions contributing to star activity: • high enzyme/DNA (>100 u/g) • low ionic strength (<25 mM) • high pH (>8) • substitution of Mg2+ • high glycerol (> 5%) • organic solvents

  7. Frequency of Restriction Sites • restriction sites ~random within genome • estimate number of sites from base composition and genome size: • at 50% GC content: • G A A T T C • (¼)(¼)(¼)(¼)(¼)(¼) = 1/4096 • if genome = 4 x 106, then 1000 sites • random distribution of sites results in fragments of various sizes

  8. Gel Electrophoresis • nucleic acids have uniform negative charge (PO4 backbone) • migration inversely related to size • structural affects • linear vs. circular • double vs. single stranded • agarose or acrylamide gels

  9. Horizontal Agarose Gel Electrophoresis • pour gel • sample preparation depends on application • electrophoresis (constant voltage) • detect with fluorescent dye (eg., ethidium bromide, SYBR, etc)

  10. 4.5 4.0 3.5 log(bp) 3.0 2.5 0.2 0.4 0.6 0.8 1 mobility Size Calculation • plot relative mobility against log of size (base pairs) • works well for linear dsDNA

  11. Secondary Structures • RNA & ssDNA form 2o structures • electrophoresis under denaturing conditions • i.e., break H-bonds • eg., urea, formamide, formaldehyde

  12. Circular vs. Linear DNA • linear DNA and circular DNA exhibit different mobilities in gel electrophoresis

  13. Circular DNA • multiple forms of circular DNA • mobility depends on size, shape and conditions

  14. Recovery of DNA from Gels • transfer to membrane (blotting) • excise band from gel • electroelution into dialysis bag • low-melting temperature agarose • dissolve gel in NaI • recover DNA • extract and precipitate DNA • adsorb DNA to silica

  15. Problems with Large DNA Molecules • difficult to handle agarose gels < 0.7% • large DNA (>10-20kb) migrates via ‘reptation’ • reptation results in similar mobilities for large molecules

  16. Pulse Field Gel Electrophoresis (PFGE) Electrode configuration of CHEF (contoured-clamp homogeneous electric field) apparatus

  17. direction of electric fields alternated at defined intervals • separation based on ability of DNA to change direction • small molecules reorient faster • up to 10 Mb can be resolved • chromosomes of lower eukaryotes • long-range restriction maps • in situ lysis of cells and restriction digests

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