Recombinant DNA Flashcards
1
Q
Definition of Cloning
A
The isolation of particular nucleotide DNA sequence from its genomic context and the production of multiple identical copies of that sequence
2
Q
Uses of Cloning (3)
A
- Isolation of genes and other DNA sequences for in vitro study
- To obtain large quantities of protein encoded by a particular gene for in vitro study (structure/fxn) or medical purposes
- Preparation of modified versions of genes for reintroduction into the original host for functional studies
3
Q
3 features of plasmid vectors
A
- origin of replication (ori site)
- selectable markers-cells carrying the plasmid need to be distinguished from those that lack the plasmid
- unique restriction enzyme cleavage sites-for the insertion of DNA sequences that are to be cloned , located in a polylinker (MCS)
4
Q
Bacteriophage cloning vectors
A
- not all bacteriophage lambda genome is essential for infection, insert foreign DNA into this region
- 20 kb fragments can be cloned
5
Q
BACs
A
- bacterial artificial chromosomes
- are simply plasmids designed to propagate large DNA fragments
- propagated at low copy number in E.coli
- 100-300+ kb fragments can be maintained
6
Q
YACs
A
- yeast artificial chromosomes
- linear DNA fragments that can be replicated in yeast
- contain yeast replication origin (ori), centromere (CEN), telomeres (TEL)
- to clone fragments up to 2000 kb
7
Q
Pulsed field gel electrophoresis (PFGE)
A
- gel is subjected to electrical fields alternating b/w different angles, allows for separation of very large DNA molecules as well as intact chromosomes
- essential for prep of inserts for BACs and YACs
- during continuous field gel electrophoresis, DNA fragments above 30-50 kb migrate w/ the same mobility regardless of size
- fragments are forced to change direction when the electrical field is reoriented
- with each reorientation, smaller sized DNA fragments begin to move in the new direction more quickly than the larger ones
- in order to isolate intact chromosomes, cells are embedded w/ low melt agarose and digested w/ a protease (proteinase K) to remove proteins (those attached to DNA like histones)
8
Q
PFGE Parameters to vary (5)
A
- agarose concentration
- field strength (volts/cm)
- run time
- pulse time (seconds/min)
- reorientation angle (usually 120 degrees)
9
Q
Directional Cloning
A
- sometimes insert orientation matters (e.g. when using an expression vector, used to make protein)
- directional cloning allows you to control the way your DNA fragment goes into the vector
10
Q
TA cloning
A
- many DNA pol add a single 3’A to blunt ended DNA via terminal transferase activity
- PCR products/A-tailed products can be ligated into T tailed vector (w/ 5’T overhangs)
11
Q
Screening for Transformants
A
pUC8 plasmid
- ampicillin resistance gene
- lacZ, codes for part of betagalactosidase enzyme
- MCS within the lacZ ORF
- transformed cells plated on antibiotic and X-gal
- beta galactosidase splits X-gal into one of its component sugars, one of which is blue
- cells containing pUC8 w/o insert are blue, those with inserted DNA are white (blue-white screening)
12
Q
Genomic Library
A
- a collection of clones that contains at least 1 copy of every DNA sequence in a genome
- in order to study a particular fragment of DNA, a clone containing that DNA can be isolated from the lib using a specific probe
13
Q
cDNA Library
A
- a collection of clones representing at least 1 copy of each of the genes expressed in a cell
- mRNA is isolated from eukaryotic cells and cDNA is synthesized by RT using the mRNA as a template (RdDp)
- reflects gene activity at the time the mRNA was isolated
14
Q
Expression Library
A
-cDNAs inserted into an expression vector (inserted genes are transcribed and translated into protein inside a host cell)
15
Q
cDNA synthesis
A
- Add an oligo (dT) primer
- first strand synthesis by RT
- now have an RNA/DNA hybrid, do a time restricted digestion so RNA serves as a template - Ribonuclease H degrades most of the RNA
- 2nd strand synthesis by DNA pol 1
- completion of 2nd strand synthesis