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Monday, 26 November 2007

genetic engineering (11) DNA preparation for cloning

DNA preparation for cloning from mRNA
1.see for abundance of mRNA -partial answer about function
eg> young RBC - large amount of Hemoglobin mRNA, chicken fallopian tube - ovalbumin mRNA 100000 molecule/cell( in contrast others species (12507) toally less than 100000
2.do not need post transcriptional modification machine of eukaryotes

Method
1. mRNA--> reverse transcriptase --> cDNA (complementary DNA)

Primers: oligo dT --> digest RNA with alkaline
sscDNA have a hook use as primer for DNA polymerase --> ds cDNA with hair pin end
S1 nuclease--> two blunt end

primer for this second strand is not so effective and S1 nuclease can shorten or unequal cut

2. use RNAse h replace alkaline --> random digest so RNA can be use as primer
second strand production by DNA polymerase I--> T4 DNA polymerase cut to be blunt end

DNA preparation by chemical synthesis
up to 50 nt
oligonucleotides and link with DNA ligase
eg: interferon gene : 66 oligonucleotides to 514 bp
commonly use for probe, primer, linker synthesis

Method
1. Phosphate triester
add protective group at amino group of A, C (benzoyl), G (isobutyryl)
add protective roup at 5' with dimethoxytrityl chloride (CH3O)2Tr-
add p-chlorophenylphosphorodichloride at 3' to link with another nucleotide [with 3' protected (berta cyanoethanol) and dimethoxytrityl at 5' removal with benzebnesulfonic]
react with triisopropylbenzenesulfonyl chloride
--> all protected dinuleotide--> select removal to control direction of synthesis

can be automated when attached with solid phase
10-20 nt in 2-3 days
2. Phosphite triester
linker is nucleoside 3- phosphoramidite
different protected group and removers
15 min 50 bp good quality

genetic engineering (10)DNA preparation for cloning

DNa preparation from cell
keep Easy, Feasible, Simple

Animal cells:
Liver, spleen,kidney, blood, cell culture
Liver: fast 24 hr to decrease glycogen
Spleen: good source, large amount

fresh tissue or frozen tissue (-80 can keep for 1 year)

Principles see isolation DNA protocol
1. Cell lysis by detergent: SDS, sarkosyl, proteinase K
2. Extract protein and cell debris with phenol
3. Pecipitate DNA with ethanol, isopropanol
3. Purification by high g centrifugation in CsCl, ethidium bromide
Electrophoresis to measure size , quantitate with OD method

Plant cells : ask botanist I don't know much about these.

genetic engineering (9)

interest DNA: genomic DNA, complementary DNA chemical or enzymatic synthetic

+vector : plasmid,phage, cosmid, phagemid, BAC, YAC

=recombinant DNA-->host: bacteria, yeast, fungi, animal cells, plant cells

genetic engineering (8)

DNA modifying enzymes
T4 polynucleotide kinase-T4 Ecoli ATP at 5' or exchange reaction-5' labeling*****
Alkaline phosphatase-bacterial AP(BAP-heat satble) or calf intestinal AP (CIP-heat labile)-5' digest Phosphate group ,prevent ligase action
DNA ligase-E coli ligase (NAD) or T4DNA ligase (ATP) nick repair or link cohesive end or blunt end (T4 ligase)
Terminal deoxynucleotidyl transferase-bovine thymus-3' add dNTP without template add complementary sequence at one end of vector and one end of gene


Nuclease
NUclease Bal 31-Alteromonas espejiana Bal 31 5'->3' exonuclease , ssDNA endonuclease
Exonuclease III-E coli3'->5' exonuclease (cannot use 3' protruding)
S1 Nuclease-Aspergillus oryzae-endonuclease ssDNA, ssRNA, nick or gap in duplex DNA, or RNA use to detect non-complete complementary, produce blut end, hairpin cDNA break
Mung beab nuclease (MB nuclease)-Mung bean-ssDNA, ssRNA, gap in duplex DNA, or RNA
DNAse I- cow pancreas- endonuclease ss,ds, with Mg - random, with Mn-blunt end or 1-2 bp protruding end
RNase -E coli digest RNA hybrid with DNA

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)