genetic engineering (6)

Enzymes for cloning
1. restriction enzyme or restriction endonulease
defense mechanisms of bacteria: restriction system, modification system (methylation)
Type
Type 2 is the enzyme use in genetic engineering
single polypeptide
cleave site in or near restriction recognition
need only Magnesium ion
restriction only
Type 1
3 polypeptides
DNAse, methyla
specific recognition site, but not the cleave site (far 400-7000 bp)
need Mg, ATP, SAM
no function after nuclease
Type 3
2 polypeptides
DNAse, methylase
cleave about 25-27 b from RS
need Mg, ATP

Type 2 naming Italic font
First letter -capital letter, genus
SEcond and third-small letter, species
strain-optional
Roman number-from discovery

RS - 4-6 bp -axis of symmetry, palindrome(not necessary)
sticky or cohesive end
5' protruding or 3" protruding
blunt or flush end
isoschizomer-same RS, not necessary same cleave site
90 RS

probabilties to find RS 4 bp= 25 bp
probabilities to find RS 6 bp= 4096 bp

conditions:
Tris HCl
Mg
NaCl
2-mercaptoethanol
pH 7.2-7.6
37 degree celcius
restrict to commercial recommendation to avoid star activity!
enzyme usually in glycerol so avoid too much enzyme

restriction map
-restriction enzymes
-DNA gel electrophoresis
polyacrylamide- 6 bp (20% acrylamide)-1000 bp (3% acrylamide)
agarose-70 bp (3%)-80000 bp (0.1%)
-migrate inverse log of bp
-visulaize-autoradiograph, ethidium bromide

Method 1- partial digestion of 1 enzyme
Method 2-complete digestion of more than 1 enzyme

Southern blot
-transfer to membrane (nitocellulose, nylon)
-denature to siigle stranded
-hybridization with DNA probe-autoradiograph

Northern blot- RNA
Western blot- protein

mutation can create or delete RS lead to change of restrction pattern-restriction fragment length polymorphism (RFLP)

genetic engineering (5)

RA synthesis
5'->3' direction
no primer
DNA dependent RNA polymerase (RNA polymerase)
ATP, GTP, CTP, UTP
anticoding strand, template strand


promotor -RNA polymerase (alpha, beta, delta)binding site - 5' ustream to transcription initiation site (+1)
termination -Rho protein, inverted repeat
prokaryote - -35 and - 10 consensus sequence- Pribnow box
Eukaryote -75 CAAT box -5'GGNCAATCT3', -25 TATA box,Hogness box - 5' TATAAA3'
higher organisms
RNA pol 1 polA-rRNA except 5 SrRNA, super repeat promotor
RNA pol2 polB-mRNA- normal promotor, AAUAAA-polyadenylation signal (20-30 b before 3')
RNA pol3 polC-tRNA, 5srRNA, scRNA, internal control region (ICR)

Posttranscriptional modification
tRNA- methylation, oxygen-->sulfur
rRNA-methylation, shortening
mRNA
-7 methyl guanosine capping
-poly Adenylation tail
-splicing -exclude intron-snRNPs, some self splicing

alpha interferon - no intron
beta globin -2 intron
factor VIII-25 intron
human thyroglobulin- > 40 intron