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

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

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 EcoRI methylase

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

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

Practical Considerations Conditions contributing to star activity: high enzyme/DNA (>100 u/ g) low ionic strength (<25 mM) high pH (>8) substitution of Mg 2+ high glycerol (> 5%) organic solvents 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

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 10 6, then 1000 sites random distribution of sites results in fragments of various sizes

Gel Electrophoresis nucleic acids have uniform negative charge (PO 4 backbone) migration inversely related to size structural affects linear vs. circular double vs. single stranded agarose or acrylamide gels

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

Size Calculation mobility log(bp ) plot relative mobility against log of size (base pairs) works well for linear dsDNA

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

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

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

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

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

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

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