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)

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

• 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

Classes of Restriction Enzymes

Type Icleavage occurs 400-7000 bpfrom recognition site

Type IIcleavage occurs adjacent orwithin recognition site

Type IIIcleavage occurs 25-27 bpfrom recognition site

• 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’-PO4

and 3’-OH• both strands cleaved

between same residues: • blunt ends• 5’-overhangs• 3’-overhangs

Enzyme Site

ClaIA T C G A TT A G C T A

EcoRIG A A T T CC T T A A G

FnuAIG A N T CC T N A G

HaeIIIG G C CC C G G

HindIIG T Y R A CC A R Y T G

HindIIIA A G C T TT T C G A A

PstIC T G C A GG A C G T C

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 CCCGGG

• Xma I CCCGGG

Compatible Ends• Pst I CTGCAG• Nsi I ATGCAT

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

• 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 106, then 1000 sites • random distribution of sites results in

fragments of various sizes

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

%agarose

range(kb)

%acrylamide

range(bp)

0.7 0.8-10 3.5 100-10000.9 0.5-7 5.0 80-5001.2 0.4-6 8.0 60-4001.5 0.2-4 12.0 40-2002.0 0.1-3 20.0 10-100

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

2.5

3.0

3.5

4.0

4.5

0.2 0.4 0.6 0.8 1mobility

log

(bp

)

• plot relative mobility against log of size (base pairs)

• works well for linear dsDNA

• RNA & ssDNA form 2o structures

• electrophoresis under denaturing conditions• i.e., break H-bonds• eg., urea, formamide,

formaldehyde

SecondaryStructures

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