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

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2
Q

Uses of Cloning (3)

A
  1. Isolation of genes and other DNA sequences for in vitro study
  2. To obtain large quantities of protein encoded by a particular gene for in vitro study (structure/fxn) or medical purposes
  3. Preparation of modified versions of genes for reintroduction into the original host for functional studies
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3
Q

3 features of plasmid vectors

A
  1. origin of replication (ori site)
  2. selectable markers-cells carrying the plasmid need to be distinguished from those that lack the plasmid
  3. unique restriction enzyme cleavage sites-for the insertion of DNA sequences that are to be cloned , located in a polylinker (MCS)
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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
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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
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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
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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)
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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)
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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
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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)
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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)
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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
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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
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14
Q

Expression Library

A

-cDNAs inserted into an expression vector (inserted genes are transcribed and translated into protein inside a host cell)

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15
Q

cDNA synthesis

A
  1. Add an oligo (dT) primer
  2. first strand synthesis by RT
    - now have an RNA/DNA hybrid, do a time restricted digestion so RNA serves as a template
  3. Ribonuclease H degrades most of the RNA
  4. 2nd strand synthesis by DNA pol 1
  5. completion of 2nd strand synthesis
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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
  1. agar plate w/ transformed bacterial colonies
  2. press nitrocellulose onto agar plate, some cells from each colony stick to the paper
  3. treat w/ alkali to disrupt cells and expose denatured DNA
  4. Incubate the paper w/ the radio-labeled DNA probe, then wash
  5. 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