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Gene Isolation and Manipulation
Terms in this set (29)
sample of DNA containing gene of interest
plasmids or bacterial viruses that are designed to carry and amplify a gene of interest
How is the donor DNA sequence cut up?
use of restriction enzymes
Restriction enzymes are based on....
the fact that any DNA sequence will carry the restriction site by just chance
the sticky ends of both strands have the same sequence in antiparrallel orientation
single stranded pairing of recombinant DNA with single stranded plasmid
Restriction enzymes cleave
Phosophodiester bonds (phosphate-carbon bonds or the backbone)
a specialized DNA polymerase from a bacteria that lives in areas of high heat, needed for PCR rxns
the DNA version of an mRNA molecule
How is cDNA produced?
By the use of reverse transcriptase and the mRNA in question
What type of primer is used in making cDNA?
poly T tail b/c it will bind to the poly A tail
Purpose of using the same restriction enzyme on both the plasmid and desired sequence?
gives us sticky ends that will conplement each other on both strands of DNA
Purpose of DNA ligase
joins the backbone of the recombinant DNA molecule with the plasmid
How can we clone blunt end strands?
Add short double stranded nucleotides that contain a restriction site and treat as normal
Each bacterial cell contains ____ copies of plasmids
All vectors must have
one restriction site where the cloned DNA needs to be inserted
Problem with using plasmid vectors
the DNA we want to be cloned must be taken up via transformation, no guarentee that it will be taken up
Solution for using plasmid vectors
Can a pUC18 plasmid vector, insert the DNA to be cloned in the gene for B-galactasidase, and plate on the agar. The bacteria that can produce B-galactasidase will break down the X-Gal sugar in the agar and produce a blue hue while those that cant will make a white hue
Bacteriophage lambda is useful for
cloning double stranded DNA as long as 15kb (the limit is 50kb but most of that codes the genes necessary for the phage to operate)
sticky ends of the bacteriophage genome
vectors that can carry 35-45 kb inserts, made from phages and act as syinges
Most popular vector for cloning is ____
BAC is derived from and
F plasmid, can hold 100-200 kb and used in whole genome sequencing
How are cells made competent
Incubation in calcium solution or shocking with electrocity, causes the membrane to accept DNA more readily
Fosmids are introduced by _____ and result in ____-
transduction, more bacteria
Phages are introduced by _______ and result in _____
infection, phage plaques
genome libraries require
numerous clones to ensure that all sequences are accounted for
cDNA libraries represent
only the protein coding regions of the genome
If a linear DNA molecule has n sites for restriction enzymes, how many pieces will be left
n + 1
THIS SET IS OFTEN IN FOLDERS WITH...
Gene regulation in bacteria
Gene reg. in eukaryotes
Chapter 10--Gene Isolation and Manipulation
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