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
16
Q
Construction of Libraries
A
- vectors used for libraries can be plasmids, BACs, bacteriophage lambda, etc (lib insert can be large or small)
- starting material needs to be prepared so it is compatible with the vector:
- restriction enzyme digestion of DNA, ligation of inserts to vector
- random sheering of DNA, blunt end ligation
- addition of linkers to DNA that are compatible w/ vector
- goal is to produce a lib of recombinant molecules as complete as possible
17
Q
Screening-RNA/DNA probes to screen libraries by hybridization
A
- colony hybridization
- plaque hybridization (bacteriophage lib)
18
Q
Screening-can use antibodies to screen for expression libraries
A
- allows you to isolate cDNAs when you don’t have a suitable DNA/RNA probe
- antibody reacts w/ expressed protein to identify clone of interest
19
Q
Colony Hybridization
A
- agar plate w/ transformed bacterial colonies
- press nitrocellulose onto agar plate, some cells from each colony stick to the paper
- treat w/ alkali to disrupt cells and expose denatured DNA
- Incubate the paper w/ the radio-labeled DNA probe, then wash
- probe annealed w/ colonies of interest, expose x ray film to paper
- go back to original agar plate, pick colonies, grow in liquid culture, isolate plasmids/BACs
20
Q
Chromosome walking
A
- classical technique used to construct a contig: a contiguous set of overlapping DNA sequences
- starting w/ the DNA sequence of a single clone, probes can be used to screen a genomic library for additional clones that contain fragments overlapping w/ the original clone
21
Q
PCR
A
The procedure uses:
- a thermostable DNA polymerase
- a pair of oligonucleotides (primers)
- a small amnt of target DNA
- dNTPs
- a repeated series of pol-catalyzed DNA synthesis rxns
22
Q
Uses of PCR (4)
A
- molecular biology
- medical diagnostics
- forensic sciences
- environmental micro, molecular ecology, molecular anthropology
23
Q
PCR is a 3 step process
A
Denaturation-denature the target sequence into ss DNA (90-94)
Annealing-anneal the oligo primers to ss template DNA (depends on primers, 45-60)
Extension-synthesize complementary strands (depends on pol, Taq=72)
24
Q
Molecular Biology Applications of PCR
A
- probe synthesis (perform PCR in the presence of labelled molecule)
- cloning add restriction sites to the 5’ end of PCR primers, amplified product can digest to produce vector compatible ends
25
Q
Problems w/ PCR
A
- DNA synthesis by Taq is sloppy (lacks proofreading), errors can occur
- method is sensitive, care must be taken to avoid contamination
- requires knowledge of the target region of DNA, so oligos can be designed
26
Q
RT-PCR (reverse transcriptase)
A
- synthesize cDNA from RNA using RT
- use cDNA as template for PCR
27
Q
qPCR (quantitative)
A
- used to quantify the abundance of a particular DNA sequence in a sample
- uses fluorescence to measure amplification of target as it happens
- often combined w/ RT-pcr to study differences in gene expression from sample to sample
