prepare foreign target DNA - PowerPoint PPT Presentation

1 / 16
About This Presentation
Title:

prepare foreign target DNA

Description:

ligate foreign DNA and vector. introduce vector into host. screen ... DNA ... DNA Ligase. Intramolecular vs. Intermolecular. Removal of 5'-PO4 Prevents ... – PowerPoint PPT presentation

Number of Views:58
Avg rating:3.0/5.0
Slides: 17
Provided by: MarkF4
Category:
Tags: dna | dna | foreign | prepare | target

less

Transcript and Presenter's Notes

Title: prepare foreign target DNA


1
Recombinant DNA
  • prepare foreign (target) DNA
  • prepare vector (host)
  • recombine target and vector DNA
  • introduce rDNA to host
  • screen for DNA of interest

2
gDNA vs cDNA
  • level of expression
  • differential gene expression
  • accessibility
  • introns
  • complicates gDNA analysis
  • can preclude expression
  • ease of preparation
  • cDNA more work

3
Preparation of cDNA
  • 1) Isolate mRNA
  • 2) Synthesize DNA-RNA hybrid
  • reverse transcriptase
  • oligo-dT primer
  • random priming
  • 3) Synthesize 2nd DNA strand
  • 4) Add termini

? RNA dependent DNA polymerase
4
  • 1) Isolate mRNA
  • 2) Synthesize DNA-RNA hybrid
  • 3) Synthesize 2nd DNA strand
  • self-priming
  • replacement synthesis
  • primed synthesis
  • 4) Add termini

5
  • 1) Isolate mRNA
  • 2) Synthesize DNA-RNA hybrid
  • 3) Synthesize 2nd DNA strand
  • 4) Add termini
  • i.e., linkers

CGGAATTCCG GCCTTAAGGC Eco RI
6
Recombinant DNA Vectors
  • autonomously-replicating DNA used to carry and
    amplify foreign DNA within host cell
  • eg plasmids, phage/viruses, or combinations
  • Plasmids
  • extra-chromosomal elements
  • 1-200 kb size range
  • transmitted during conjugation
  • antibiotic resistance
  • low copy number vs high copy number

7
Useful Plasmid Features
  • Relaxed Replication
  • Selectable Markers
  • Streamlined
  • Polylinker or MCS
  • Identification of Recombinants
  • most derived from pUC or pBR322

8
Generic rDNA Protocol
  • prepare foreign DNA
  • prepare vector
  • ligate foreign DNA and vector
  • introduce vector into host
  • screen for rDNA of interest

9
Ligation Reaction
  • mix foreign and vector DNA in presence of DNA
    ligase
  • optimal ratios of vector to insert generally
    1.5-21
  • intermolecular base-pairing can occur between
    compatible overhangs

10
DNA Ligase
  • catalyzes the formation of phosphodiester bond
    between 5-PO4 and 3-OH
  • i.e., joins DNA fragments
  • typically carried out at lower temperatures
    (8-16o) for extended periods

11
Intramolecular vs. Intermolecular
12
Removal of 5-PO4 Prevents Vector Self Ligation
13
(No Transcript)
14
  • prepare foreign DNA
  • prepare vector
  • ligate target and vector
  • introduce rDNA to host
  • heat shock Ca2
  • electroporation
  • select for transformants with antibiotic
  • screen for rDNA of interest

15
Colony Lift
  • Sources of Probes
  • cloned genes
  • synthetic oligonucleotides
  • PCR products

16
Identifying Recombinants
  • based on interruption of a gene
  • eg., lacZ gene b-galactosidase
  • intact b-galactosidase produces blue color in
    presence of X-gal
  • ?-complementation or blue-white screening
Write a Comment
User Comments (0)
About PowerShow.com