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Sunday, 25 November 2007

Genetic engineering (2)

Structure and function of Deoxyribonucleic Acid (DNA)
Structure and behavior of DNA and RNA (Ribonucleic acid)
Chemical composition
Nucleotide=Base+Sugar+Phosphate group
Nucleotide=(Purine: adenine(A), guanine(G) or Pyrimidine: Cytosine(C), Thymidine (T-DNA only), uracil (U-RNA only)+ Deoxyribose (DNA) or Ribose(RNA)
5' --> 3 '
Structure of DNA
B-DNA
double helix
antiparallel
base -hydrogen bone- base
A=T, G-triple bond-C in horizontal plane
sugar back bone
phosphate outside
3.4 nm height for each round 10 base
diameter 2 nm
A-DNA
relative humidity 75%, Sodium, Potassium, or Cesium
base diagonal 20 degree, each base differ 0.26 nm
11-12 bp each round
diameter 2.3 nm
antiparallel
RNA, or DNA-RNA hybrid
Z-DNA
poly dGC,polydAC
each base differ 0.37 nm
12 bp per each round
diameter 1.8 nm
Zigzag-like
stabilized with replace C in poly dGC with 5-methyl C
Denature (melting)and renature(association, annealing)
melting temperature (Tm) -temperature that have equal ssDNA and dsDNA
pHm-pH that have equal ssDNA and dsDNA
urea and formamide-decrease Tm
monovalent or divalent cation concentration-increase Tm
G+C/A+T increase Tm. pHm
GC increase 1%, Tm increase 0.4 degree
renaturation between different species of nucleic acid-hybridization: solution, filter
Other qualities of DNA
1. UV absorption: absorbance (A) or optical density (OD) peak at 260
OD of 1 mg/ml of DNA, RNAA, oligonucleotide = 20, 25, 30
OD increase with temperature (ssDNA-> increase OD)
2. Acid-base:stable at pH 4-5, depurination at pH <3, base donot react with DNA, but destroy phosphodiester bond of RNA (react at OH)
3.sedimentation
velocity sedimentation: generate gradient column of sucrose or potassium citrate-add DNA mixture-centrifuge-highMW down, low MW up
equilibrium sedimentation:CS-->spin down centrifuge-->diffusion until equilibrium-add Mixture buoncy of DNA=Cs, protein-top, RNA-bottom

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