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

Genetic engineering (1)

Introduction
Basic genetics to genetic engineering
1866 Mendel - Principle of Inheritance
1900 de Vries, Correns, van Tschermak - rediscover Mendel's rule
1905 Bateson - word "Genetics"
1901 Johannsen - word "Genes"
1928 Avery, Macleod, McCarthy- DNA is genetic material
1953 Watson, Crick - double helix DNA
Beginning of genetic engineering techniques
1970 Nathans, Smith - restriction enzymes
1970 reverse transcriptase
1976 antibody diversities
1977 split genes: introns and exons
1977 Sequencing techniques
1982 Supermice: Growth Hormone Gene Transfer
1983 Ribozymes
1985 Polymerase chain reaction (PCR), DNA fingerprint
1990 CFTR gene for cystic fibrosis
1993 IT15 gene for Huntington disease
1997 Telomerase gene
2003 First draft of Human Genome