* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download 2 - cellbiochem.ca
Human genome wikipedia , lookup
DNA profiling wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
Whole genome sequencing wikipedia , lookup
DNA polymerase wikipedia , lookup
Nutriepigenomics wikipedia , lookup
SNP genotyping wikipedia , lookup
Primary transcript wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Genealogical DNA test wikipedia , lookup
DNA sequencing wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
Designer baby wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Genetic engineering wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
Point mutation wikipedia , lookup
Microsatellite wikipedia , lookup
Non-coding DNA wikipedia , lookup
Genome editing wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Microevolution wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
DNA supercoil wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Epigenomics wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
DNA vaccination wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Metagenomics wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Helitron (biology) wikipedia , lookup
Molecular cloning wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Recombinant DNA Technology CHMI 4226 E Week 2 12 January 2009 Toolbox part 2. Plasmids, DNA cloning 101, DNA sequencing E.R. Gauthier, Ph.D. CHMI 4226 F 1 Plasmids • Plasmids: – Extrachromosomal (episomal) circular DNA molecules – Minimal features: • Origin of replication: allow the plasmid to replicate autonomously • Antibiotic resistance gene: allow for selection for bacterial cells that have taken up the vector • Multiple cloning site (MCS): a small region of the plasmid engineered to contain the cleavage sites for a limited number of restriction enzymes E.R. Gauthier, Ph.D. CHMI 4226 F 2 Plasmids – origin of replication • Origin of replication (Ori): small DNA sequences allowing the bacterial DNA polymerase to bind and initiate DNA replication; • Several Ori can be found. ColE1 is the most frequently encountered; • Some Ori allow the plasmid to replicate frequently (high copy number plasmids – up to 100 copies per cell); others allow only a low rate of replication initiation (low copy number plasmids – only a few copies per cell) E.R. Gauthier, Ph.D. CHMI 4226 F 3 Plasmids – selection genes • Genes which encode for proteins affording resistance to a specific antiibotic; • Most frequently encountered: – Ampr: resistance to ampicillin – Kanr: resistance to kanamycin – Tetr: resistance to tetracyclin • Bacteria possessing a plasmid with the Ampr gene will survive when plated onto a media containing ampicillin; E.R. Gauthier, Ph.D. CHMI 4226 F 4 Plasmids – multiple cloning sites • MCS: a limited region on the plasmid which has been engineered to contain unique cleavage sites for a selected number of restriction enzymes; • RE cleavage sites in the MCS are not found anywhere else on the plasmid. E.R. Gauthier, Ph.D. CHMI 4226 F 5 Plasmids – other features • Promoters for viral RNA polymerases Sp6, T7 or T3 – Allows for in-vitro trasncription experiments • Origin of replication of phage F1: – Enables the production of single-stranded plasmids • Genes encoding supressor tRNAs • Reporter genes: – Allows for the easy identification of bacterial cells with desired features – E.g.: Lac z (encodes b-galactosidase) E.R. Gauthier, Ph.D. CHMI 4226 F 6 Plasmids – SupF selection gene • SupF: – Suppressor tRNA – Inserts an amino acid (Glu) at the UAA stop codon – Allows the survival of bacteria with a P3 episome : a plasmid containing the Ampr and Tetr genes both interrupted with a UAA stop codon. E.R. Gauthier, Ph.D. CHMI 4226 F 7 E.R. Gauthier, Ph.D. CHMI 4226 F 8 Plasmids – pBR322 E.R. Gauthier, Ph.D. CHMI 4226 F 9 Plasmids - pBluescript E.R. Gauthier, Ph.D. CHMI 4226 F 10 Plasmids – shuttle vectors • Shuttle vectors can be used in at least two different organisms: – – – – – Bacteria (mandatory) Yeast Insects plants mammals E.R. Gauthier, Ph.D. CHMI 4226 F 11 Plasmids – shuttle vectors • Bacteria/Yeast shuttle vector; • Leu2: – Selection marker for growth in yeast – Encodes for a gene which allows for the synthesis of the amino acid leucine in a yeast strain which cannot produce Leu on its own (auxotrophic) – Yeast with this vector can be grown on media devoid fo leucine. E.R. Gauthier, Ph.D. CHMI 4226 F 12 DNA cloning • Why cloning DNA? – To produce greater amounts of YFG – To characterise the properties of YFG • sequencing • Mutagenesis – To express YFP in vitro or in a living organism • production of recombinant insulin: much better than insulin purified from blood, which can be contaminated with viruses (hepatitis C, HIV) or other nice things (prions) E.R. Gauthier, Ph.D. CHMI 4226 F 13 DNA cloning – basic steps • 1. cut DNA of interest and plasmid with appropriate restriction enzyme • 2. mix together and seal free ends with DNA ligase (ligation) • 3. Insert DNA in bacteria (transformation) – Requires transformation-competant bacteria • 4. Select antibiotic-resistant bacteria and search for cells having the recombinant plasmid. • 5. Confirm cloning – restriction enzyme digest – DNA sequencing E.R. Gauthier, Ph.D. CHMI 4226 F 14 DNA cloning – basic steps E.R. Gauthier, Ph.D. CHMI 4226 F 15 DNA cloning • Treating vector with alcaline phosphatase – reduces background of bacteria with plasmid not containing the insert. • Blunt-ends can be used: – a greater insert DNA/plasmid ratio must be used: 10/1 instead of the usual 3/1 – Much, much less efficient than cloning with protruding ends • The ends of your DNA fragment can be modified with Klenow enzyme to accomodate the available RE sites in the vector (and vice-versa). • Adaptors and linkers can be used to facilitate cloning. E.R. Gauthier, Ph.D. CHMI 4226 F 16 Adaptors and linkers Adaptors Linkers E.R. Gauthier, Ph.D. CHMI 4226 F 17 Assignment #2 E.R. Gauthier, Ph.D. CHMI 4226 F 18 Use of b-gal - Bacteria used in genetic engineering express a portion of b-gal missing the Nterminal 50 amino acids (called the omega [w] fragment); -Several vectors carry the missing Nterminal 50 amino acids (a fragment) of bgal in the MCS; - Only the bacteria having both the a and w portion of b-gal can express a functional enzyme (a complementation) E.R. Gauthier, Ph.D. CHMI 4226 F 19 Use of b-gal • If a DNA fragment is inserted into the MCS of pBluescript, it interrupts the b-gal a fragment, making it inactive; • SO: bacteria with a recombinant plasmid will not express a functional b-gal, and will turn white upon staining wity X-gal; • However, bacteria with a plasmid which does not contain the DNA fragment will produce the a fragment, will have a functional b-gal enzyme, and will turn blue upon staining with X-gal. E.R. Gauthier, Ph.D. CHMI 4226 F 20 DNA cloning – plasmid purification from bacteria Culture 1 bacterial colony into liquid media ..... . Recover bacterial cells by centrifugation ..... . 1) Resuspend cells in glucosebased buffer 3) Precipitate bacterial chromosome and proteins with acidic buffer and centrifuge E.R. Gauthier, Ph.D. 2) Lyse bacteria with SDS/NaOH buffer CHMI 4226 F Extract supernatant with basic (pH 8) phenol - solubilizes contaminating proteins Extract with chloroform – to get rid of the phenol Precipitate plasmid DNA with ethanol Dry pellet, resuspend in buffer, digest with restriction enzyme and analyse on agarose gel 21 DNA cloning – identification of recombinant plasmids • Three important tests have to be done: – Cut with RE to see if the insert is of the correct size; – Cut with RE to confirm the identity of the fragment (usually choose RE that cut inside the fragment) – Sequence the insert. E.R. Gauthier, Ph.D. CHMI 4226 F 22 DNA sequencing E.R. Gauthier, Ph.D. CHMI 4226 F 23 DNA sequencing E.R. Gauthier, Ph.D. CHMI 4226 F 24 DNA sequencing 3’ A G T C G C A C T A G T G C A T A G 5’ E.R. Gauthier, Ph.D. CHMI 4226 F 25 DNA sequencing E.R. Gauthier, Ph.D. CHMI 4226 F 26 DNA sequencing E.R. Gauthier, Ph.D. CHMI 4226 F 27 What to do with the data? • 1. identification (did I clone the piece of DNA I wanted) • 2. Is it complete, or do did I clone only a portion of it (happens more often than you think…)? • Are there any mutations (happens a lot…)? E.R. Gauthier, Ph.D. CHMI 4226 F 28 BLAST search • Blast: algorithm that allows you to quickly identify sequences that are similar to the DNA of interest; • Blastp: similarity searches with amino acid sequences • Blastn: similarity searches with nucleotide sequences E.R. Gauthier, Ph.D. CHMI 4226 F 29 BLAST search QUERY sequence(s) BLAST results BLAST program BLAST database E.R. Gauthier, Ph.D. CHMI 4226 F 30 BLAST search E.R. Gauthier, Ph.D. CHMI 4226 F 31 BLAST search E.R. Gauthier, Ph.D. CHMI 4226 F 32 BLAST search E.R. Gauthier, Ph.D. CHMI 4226 F 33 BLAST search E.R. Gauthier, Ph.D. CHMI 4226 F 34 BLAST search E.R. Gauthier, Ph.D. CHMI 4226 F 35 BLAST search E.R. Gauthier, Ph.D. CHMI 4226 F 36 BLAST search • E value: indicate the odds that the hit is attributable to chance only. – The lower the E value, the better the odds that it is a real match. • G: link to Entrez Gene • U: link to sequencerelated info (expression profile, chromosomal location, orthologs) • E: link to GEO (Gene Expression Omnibus): database of gene expression profiles. E.R. Gauthier, Ph.D. CHMI 4226 F 37 BLAST search E.R. Gauthier, Ph.D. CHMI 4226 F 38 BLAST search E.R. Gauthier, Ph.D. CHMI 4226 F 39 Translation of sequencing data • Allows you you determine if the sequence you isolated contains the entire open reading frame. – Open reading frame (ORF): • the nucleotide sequence coding for a protein; • Starts with a AUG codon, and stops with one of the 3 stop codons (UAA, UAG, UGA). E.R. Gauthier, Ph.D. CHMI 4226 F 40 BLAST search E.R. Gauthier, Ph.D. CHMI 4226 F 41 Translation of sequencing data E.R. Gauthier, Ph.D. CHMI 4226 F 42 Translation of sequencing data E.R. Gauthier, Ph.D. CHMI 4226 F 43 Translation of sequencing data E.R. Gauthier, Ph.D. CHMI 4226 F 44 Translation of sequencing data E.R. Gauthier, Ph.D. CHMI 4226 F 45 Translation of sequencing data E.R. Gauthier, Ph.D. CHMI 4226 F 46 Translation of sequencing data E.R. Gauthier, Ph.D. CHMI 4226 F 47 Blastp result E.R. Gauthier, Ph.D. CHMI 4226 F 48 Assignment #3! E.R. Gauthier, Ph.D. CHMI 4226 F 49