Genetic Engineering DannyChan

Genetic Engineering DannyChan - Genetic Engineering and...

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

View Full Document Right Arrow Icon
Genetic Engineering and Applications (BIOC 2603) Dr. Danny Chan Department of Biochemistry Email: chand@hkusua.hku.hk Phone: 28199842
Background image of page 1

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

View Full DocumentRight Arrow Icon
Topics in year 1 • Restriction enzymes. • DNA modifying enzymes. • Cloning using plasmids as vectors. • cDNA library. • Screening libraries. • Methods of labeling DNA. • DNA sequencing
Background image of page 2
Topics in year 2: • Other cloning systems – Phages, cosmids, BACs and YACs • Expression vectors – Prokaryote and eukaryote • Applications • Polymerase chain reaction
Background image of page 3

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

View Full DocumentRight Arrow Icon
TISSUE mRNA DNA gene function gene structure & homology gene expression cDNA DNA fragments DNA sequence (bioinformatics) DNA clones gene regulation probes gene targeting in mice amino acid sequence protein structure & homology biosynthesis linkage analysis genetic diseases therapeutic treatments industrial applications medical products gene location
Background image of page 4
Theme 1 Other cloning systems • Phage vectors • Phagemids • Cosmids •B a c s •Y a c s Analysis of whole genomes
Background image of page 5

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

View Full DocumentRight Arrow Icon
Cloning vectors • Plasmids: < 10 Kb • Bacteriophage λ : 2-25 Kb • Cosmids: 35-50 kb • Bacteria artificial chromosomes (BACs): 50-300 Kb • Yeast artificial chromosomes (YACs): 100-2000 Kb
Background image of page 6
DNA Protein coat Bacteriophage λ Linear, double-stranded phage, ~ 49 kb. Process of infection: phage particle injects its linear DNA into the cell, where it is ligated into a circle. replicated by forming more phage particles, release from the cell by lysis and cell death ( lytic phase ), or the DNA may intergrate with the host genome by site-specific recombination, where it may remain for a long time ( lysogenic phase )
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
Bacteriophage vectors The infection and subsequent lysis of E. coli by bacteriophage λ may be used to propagate cloned DNA. Commonly used in generating genomic DNA libraries. Phage λ can accommodate larger DNA fragments. Non- essential portion of the linear 48.5 kb λ genome may be replaced by up to 25 kb foreign DNA can be used as a cloning vector. ~ 16 fold advantage in cloning efficiency compared with plasmids.
Background image of page 9

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

View Full DocumentRight Arrow Icon
λ Genome 48.5 kb long arm (left) short arm (right) cos cos Non-essential 5’ -CGGGGCGGCGACCTCG- 3’ 3’ -GCCCCGCCGCTGGAGC- 3’ 5’ -CG 3’ -GCCCCGCCGCTGGA GGGCGGCGACCTCG- 3’ GC- 3’ Ligation (after infection) Cleavage (during packaging) + Cos site
Background image of page 10
λ Insertion and λ replacement vectors • Insertion vectors accept DNA directly into the “stuffer” region. • replacement vectors replace the stuffer sequence with foreign DNA. • Insertion vectors accept less DNA than replacement vectors.
Background image of page 11

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

View Full DocumentRight Arrow Icon
Cloning into a λ insertion vector left arm right arm E E cos cos left arm right arm E cos cos Digest with restriction enzyme E E Target DNA + DNA ligase Infect E.coli cells cos cos cos Insert 1 Insert 2 R L L R E E E E Package with λ extract
Background image of page 12
Cloning into a λ replacement vector left arm right arm E E cos cos left arm right arm E cos cos Digest with restriction enzymes E E Target DNA + DNA ligase E Remove stuffer fragment Too small to package cos cos cos cos Insert 1 Insert 2 L R L L RR E E E E E Package with λ extract Infect E.coli cells
Background image of page 13

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

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

This note was uploaded on 03/19/2010 for the course BIOC BIOC2603 taught by Professor Dr.mhsham during the Spring '07 term at HKU.

Page1 / 48

Genetic Engineering DannyChan - Genetic Engineering and...

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