Genetic engineering (7)

Polymerase and exonuclease
E coli DNA polymerase I
1 chain polypeptide
109000 Dalton
5'->3' polymerase
5'->3" exonuclease
3'-> 5" exonuclease
proof reading and repair DNA
nick translation
DNA labeling- low concentration of DNAse, add label

Klenow fragment,large fragment
1 chain polypeptide
Pol I--> typsin, subtilisin 76000 Dalton
C-terminal, no 5'->3' exonuclease
use for DNA synthesis from RNA, enzymatic sequencing, add nucleotide for 5 protruding end, replacement or exchange reaction of 3' blut end

T4 DNA polymerase
same as Klenow activity, same use
but 3'->5' activity 200X
dNTP concentration control polymerase activity

RNA dependent DNA polymerase, reverse transcriptase
RNA virus enzyme
5'->3' DNA synthesis from RNA template
need ssRNA, or ssDNA as template and primer
make cDNA from mRNA, 3' fill in

Polymerase Chain Reaction (PCR)

• Template with known at least head or end sequence
• Design small oligonucletide complement with 3' of each end (20-35 b)
• Mix large amount of primers with template DNA
• Deanneal template with heat
• Reanneal of primer to template
• DNA polymerase will extend DNA 5'->3'
• double each round

Taq DNA polymerase - heat stable

usually temperature setting

deanneal-95 1 min

anneal-60 1 min

extension-72 1 min

30-40 rounds

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)