Genetic Engineering - Amazon S3

onwardhaggardBiotechnology

Dec 12, 2012 (4 years and 11 months ago)

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BI
OPL 2400

9/10/2009 3:16:00 PM

Transforming an Organism



Find your gene of interest



Make lots of copies of your gene
-
cloning



Insert it into genome of organism



Determine if gene if functional



Regenerate organism

Genetic Engineering



Protein/mRNA


reverse transcriptase and cDNA



Reverse Transcriptase


viral enzyme makes DNA from RNA


called cDNA



Retrovirus that the genetic material is rNA

o

Take over the cell

o

Make DNA

o

Virus spreads



You don’t get the entire sequence since parts of the sequence have
been cut out (introns)



Related
Gene/ from other organism



Find your gene in a library

o

cDNA libraries



if you want to look at the functional genes



you can bypass the extra genes



collect mRNA and use reverse transcriptase



not getting exact copy of gene

o

genomic library



take genetic material
s from organism and isolate genes



cut up into fragments

Genomic Library



Cut DNA into lots of pieces


Restriction Enzymes



Insert Pieces into plasmids



Insert plasmids into bacteria



Grow Bacteria

and multiply fragments



Probe cloned fragments to find your
gene

Bacterial Genome



Bacteria have a single circular chromosome



Also contain plasmids


small circular DNA which are transferred
during conjugation


genetic recombination

Restriction Enzymes



Cut DNA with sticky ends


useful for splicing fragments toget
her



They cut and leave some base pairs exposed

o

Palindromes

o

Useful for splicing pieces of DNA

o

How you make a genomic library


Once you have our gene reinsert into bacteria and make lots of copies


amplification

Us a reporter or marker gene to identify reco
mbinant colonies

Usually antibiotic resistance or a reporter gene

Antibiotic resistance genes


included with your gene

of interest

Reporter Genes



Produce some visible gene product when present



GUS


B
-
glucuronidase


bacterial enzyme produces a product th
at
stai
n
s a blue color



Lac
-
z genes


codes for B galactosidase


also a blue color



Green fluorescent Protein (GFP) isolated from Jellyfish

Insert gene of interest into plasmid

PCR



Polymerase Chain Reaction


another method to amplify your gene



Uses a special
type of
DNA polymerase which works at high
t
emperatures to multiply DNA quickly without Bacteria



No selection needed



Takes small fragment of DNA and makes copies of it


9/10/2009 3:16:00 PM



9/10/2009 3:16:00 PM