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Biochem Abnormal Amnio Genetic Engineering


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Hind III makes blunt or sticky ends?
can be used to clone DNA fragments of what length?
10-15 kb DNA fragments
How do you select recombinant plasmids using ampR and tetR?,
cut sticky ends in the ampR, thats where DNA should insert, but tetR will still work.

plate on tet and all the no-plasmids will die

Plate on tet and amp will show the nonrecomb plasmids
bacteriophage DNA, circular or linear?
what part of the bacteriophage DNA can be replaced? what part is essential?
Linear DNA with COS sites on the end, the central portion is not essential so can be replaced with recombinant DNA
Bacteriophage gamma can be used to clone a DNA fragment how long?
15-25 kb DNA fragment
How can you detect recombinant bacteriophage?
infected cells grow slower, can see them on a bacterial lawn
What are cosmids?
combine both bacteriophage and plasmid features
oriC, selectable markers and cos sites
How large a DNA fragment can you clone with a cosmid?
up to 46 kb fragment
how do you use cosmids?
cut cosmid at EcoRI site, insert 46 kb DNA, package into phage (cause it has cos site) and select using tetR
How big is a YAC?
several hundred kb
what are YACS
a large cloning vector
how do you clone human DNA into a yac
digest genomic dna to obtain large fragments,
digest yac with ecoRI and BamHI
genomic dna and yac combine at ecorI sites
recombinant vectors can now be used to infect cells
how do you make cDNA>
isolate mRNA using oligo t's on a column, add reverse transcriptase to make DNA, add RNaseH to cut into many segments and use DNA POL I to syn complementary strand
once you have cDNA how do you make a library
USE bacteriophage

add linkers to cDNA and cut linkers with ecorI(methylate ecorI sites on cDNA so that its not cut)
join cDNA to cos sites using DNA ligase,
uptake into bacteriophage, plate bacteriophage and select for those that took recombinant dna using bacterial lawn,
Screening DNA libraries - in situ hybridization
blot bacterial colonies onto nitrcellulose paper, lyse using naoh, dna will stick to nitrocellulose, add radioactive probe to area of interest, x-ray film, black spots are key
probe formation
isolate protein of gene, work backwards from aa sequence to get DNA sequence, need 6 aa's, use tyr and met aa's cause dont have a lot of degeneracy
make all possible sequences, and then see which one sticks
expression vectors
insert foreign DNA (must be cDNA) near promoters, often make fusion protein,
fusion protein
from expression vectors, have a little bit of bact DNA along with foreign DNA
improve stability of protein
how to screen an expression library
using antibodies to id desired protein
site directed mutagenesis
clone dna frag in ds vector, isolate ss, add mutagenic oligo to ss, add DNA pol to syn the rest
what is positional cloning?
Finding adjacent genes:

finding a diseased gene even when you know nothing about the function of the gene. See which RFLP is inherited with the disease
describe chromosome walking
use genomic clone you know of near the gene of interest
make a probe from the end of it
probe the library to see if it sticks to any other pieces beside your original piece
use the second piece of genomic DNA to make another probe
probe library again etc
eventually you'll make a clone that will hit the gene of interest (need to know gene of interest seq?)
expression of proteins in e.Coli
what type of vector?
what are the down sides
expression vector must use cDNA
most often make fusion proteins
-not suitable for post translationally modified genes (bact lack these enz's)
-some proteins dont fold correctly in bact and form inclusion bodies
expression of proteins in yeast
euk, so can fold proteins correctly/ post translational modification, transformed using plasmids
shuttle vector
A vector (e.g. a plasmid) constructed in such a way that it can replicate in at least two different host species (eg a prokaryote and a eukaryote)
expression of proteins in insects
good points
high level of expression, correct folding
baculovirus vectors
cost is intermediate between bact and mammalian cells
expression in mammalian cells
4 ways to get it DNA into cells
high cost
microinject DNA into nuceus, or Ca/DNA taken up by endocytosis or electroporation, or retroviral infection
a segment of DNA which has been isolated from one organism and is introduced into a different organism. Typically the DNA is incorporated into the organism's germ line
transgenic mice
limitation to transgenic mice in terms of how many you can get
mice who take up the introduced DNA in the pronuclei

limit - only some eggs survive 30-40%
only a fraction of those are transgenice
how can you attempt to get transgenic mice by introdusing DNA?
1 inject into pronuclei
2 introduce DNA to embryonic stem cell: (advantage - stem cell can be screened before injection into blastocyst) - forms a chimeric mouse
Chimeric mouse, how do you isolate a transfected germ line?
chimeric mice grown from infected embryonic stem cells can either have germinal uptake or only somatic.
ES - brown
Blastocyst - Black
mate chimeric mouse with black females
brown offspring are the ones from the ES cells
How do you make a knockout mouse?
Fuck up DNA of interest by inserting Ab resistance in the middle

electroporate into ES

Those that do homologous recomb will have the correct promoter near it and express the antibiotic resistance using the beginning of the messed up DNA.
Expression of proteins in milk of sheep
transgene is placed under control of a promoter only found in mammary tissue

ie Tissue Plasminogen activator gene put in expression vector near beta-lactoglobulin promoter. Injected into nucleus of sheep ova. Identify transgenic progeny through PCR of tissue plasminogen
zoo blot
Zoo blot hybridization is an assay for DNA sequences that are highly conserved between species.
S.Blot using digests with DNA from different organisms
Northern blot Vs Southern Blot
Southern = DNA , detects gene rearrangements, deletions, # of copies of a gene.
Electrophorese digested DNA, nitrocellulose blot, label with probe

Northern - RNA, use cDNA as probe, determines level of mRNA and where it is expressed(which tissues)
Tail blot
PCR and S.Blot of offspring of foster mom mouse(grows embryos that were attempted to be made trangenic)
how do you determine the sex of an embryo using a single prentatal cell?
also diagnonsis of infectious disease, forensics, prenatal screening for inherited disorders

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