october_30_2008_2_slides_per_page - endonuclease EcoRI...

Info iconThis preview shows pages 1–14. Sign up to view the full content.

View Full Document Right Arrow Icon
1 Recombinant DNA technology - “practical” use of cells and DNA - Molecular Cloning : insertion of DNA fragment from one cell/organism to the replicating DNA of another type of cell Molecular Cloning - Berg, Cohen and Boyer - mutant strains of Esherichia coli lacking restriction endonucleases - development of extrachromosomal DNA (plasmids) and bacteriophage DNA - methods to alter these plasmids and bacteriophage
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
2 Cloning vectors (i) plasmids : self-replicating extrachromosomal DNA (small, circular, dsDNA) stringent replicated plasmids relaxed replicated plasmids Ideal plasmid - relaxed - small - contain identifiable marker - restriction endonuclease sites
Background image of page 2
3 (ii) Bacteriophage DNA - λ phage most common bacteriophage - large DNA,
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
4 Reverse transcriptase - synthesizes a DNA strand from a RNA template - helps create cDNA
Background image of page 4
5 Constructing recombinant DNA (i) Obtain DNA of interest to be transferred mRNA of gene to be transferred restriction endonucleases reverse transcriptase (ii) Cleave the vector with restriction
Background image of page 5

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Background image of page 6
Background image of page 7

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Background image of page 8
Background image of page 9

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Background image of page 10
Background image of page 11

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Background image of page 12
Background image of page 13

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Background image of page 14
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: endonuclease EcoRI BamHI plasmid 6 vector 5’-3’--3’-5’ 5’-3’--3’-5’ insert OH P 7 (iii) insert foreign DNA fragment (iv) transformation (v) identifying cells with hybrid DNA 8 Replicate plating DNA used to make recombinant protein Prokaryotic Expression Vector 9 Polymerase Chain Reaction (PCR)- Amplification of DNA (if part of the sequence is known)- cloning- forensics also require some knowledge of the sequence to form primers Requirement for PCR reaction:- Two primers (@ 20 bases, single stranded) complementary to flanking sequences, needs free 3’OH-DNA polymerase- dATP, dTTP, dGTP and dCTP 10 TAQ DNA polymerase Second Cycle Begins 11 TAQ DNA Polymerase Second Cycle continues > 30 cycles 12 Genetically Altered Organisms- bacteria: routine to introduce foreign DNA- eukaryotic animals: 13 Transgenic C.elegans 14 Gene disruption by Homologous Recombination...
View Full Document

Page1 / 14

october_30_2008_2_slides_per_page - endonuclease EcoRI...

This preview shows document pages 1 - 14. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online