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...
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Transcript of 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...
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