3' mRNA 5'
inverse transferase
cDNA
DNA Ploymerase
restriction enzyme
dCTP
fragment DNA.
short circular resistant AMP
short circular resistant TET
restriction enzyme
dCTP
open plasmid.
Fragment DNA + open plasmid = rDNA.
(dlm rDNA ada once more restriction enzyme use to get cloning compound.
thus 3 times restriction is use)
inverse sinthesis DNA dr mRNA
Polymerase doubled single DNA become doubled strands.
Functioning of vector:
1.accept
2.independent
3.express
4mark
5.stuggart cutting by its restriction enzyme.
screen:
host bacteria + nutrient AMP is screened by blue-white color.
thus,1.host bacteria + lac Z(vector) gives blue.
2. host bacteria + rDNA gives white color.
hence,rDNA is isolated,purified,cloning.
HOW VECTOR PRODuCED IN rDNA
1.short circular DNA bacteria independent replicated as vector.
2.vector contain at least 1 resistant antibiotics AMP/TET.
3.vector transfer into another host by transformation.
in addition :
1.foreign fragment DNA bound together dgn plasmid formed rDNA
2.rDNA introduce into another host to replicate
3.restriction enzyme used to stuggart cutted pd polindrome DNA & plasmid
produce sticke end DNA & sticky end open plasmid
gene bank = stored copy of genetics information spesis to conservative puporses.
genomic library(rDNA) = collection clones consist DNA spesis
cDNA = collection clones only coding region of genome.
HOW.
genomic library (rDNA):
1.nuclear DNA isolated dr cell cutted by restriction enzyme.
2.vector plasmid isolated dr bacteria by restriction enzyme contain at least i resistant genes.
3.fragment DNA & open plasmid produced bound together by ligase in tube formed rDNA.
4.rDNA introduced into host bacteria to replicate independently becomes transform bacteria.
5.transforme bacteri cultured in medium contain antibiotics.
6.screening to identify which colony has rDNA.
7.once identified,bacteria isolated,purified,cloned large to prroduce large amount of rDNA.
thus,collection of rDNA spesis contain base sequences cell form genomic library.
cDNA :
1.RNA spesific cell isolated as template form DNA.
2.inverse tranferase used produce single strand cDNA.
3.DNA polymerase used doubled dNA becomes double strands DNA.
4.mRNA is degraded.
1.restriction enzyme stuggart cut DNA produce sticky ends fragment DNA.
2.restriction enzyme stuggart cut vector produce sticky ends open pasmid.
3.fragment DNA & open plasmid bound together by ligase in tube produce rDNA
4.rDNA introduce into host bacteria to replicate independently formed transform bacteria.
5.transform bacteria is cultured in medium consist antibiotics.
thus,collection of coding region genome spesis formed CDNA library.
GENE THERAPY = insertion of functional genes into certain body part.
SCID is desease of deficiency ADA in infants.
decrease in ADA cause immunation does not properly functioning.
1.normal gene ADA isolated dr human cell cloned dlm vector.
2.vector contain gene ADA introduce into host weaken retrovirus.
1.amount narrow bond isolated dr human cell to be cultured.
2.retrovirus contain non-pathogenic bound togtehr dgn narrow.
3.retrovirus integrated narrow ntroduced into patient to replicate independently.
FINGERPRITNS TECHNICS = intron does not have base sequences
analysing DNA to be compared dr differe t sources as PCR methods.
DNA genome has intron which doesn't have genes becomes uniq.
1.restriction enzyme stuggart cut DNA for fragments DNA.
2.exctracted DNA transfered into nylon membrane
3.nylon membrane has alkaline to formed single strand DNA.
4.radioactive dgn specifis base sequences is probe to single DNA
5.excess fragments DNA is washed off.
6.Residue fragment DNA exposed to X-RAY produces unique DNA fingerprints differ bands.
