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.