Material for Quiz 5: Chapter 8
Standard Recombinant DNA technique: Cloning a DNA fragment into a plasmid (up to 10 kb DNA fragments) Insert = Donor DNA = Genomic DNA Vector = Plasmid = Engineered Bacterial Plasmids (pBR322, pUC19..) Restriction Enzymes = bind and cut at palindromic sequences Digest insert and vector Leaves Cohesive (sticky) ends = 5’ or 3’ overhang Leaves Blunt ends DNA Ligase = catalyzes phosphoester bond formation using energy of ATP hydrolysis 5’Phosphate + 3’Hydroxyl – Phosphate ester Transformation of E. coli Selection of transformed colonies (selectable marker on plasmid) Amplification of plasmid DNA in bacteria Screen for Recombinant Clones Insertional inactivation of gene on plasmid Antibiotic resistance (pBR322) Lac Z gene (pUC19) Analysis of Recombinant Clones Restriction mapping DNA sequencing
Standard Recombinant DNA technique: Cloning a DNA fragment into a plasmid (up to 10 kb DNA fragments) Insert = Donor DNA = Genomic DNA Vector = Plasmid = Engineered Bacterial Plasmids (pBR322, pUC19..) Restriction Enzymes = bind and cut at palindromic sequences Digest insert and vector Leaves Cohesive (sticky) ends = 5’ or 3’ overhang Leaves Blunt ends DNA Ligase = catalyzes phosphoester bond formation using energy of ATP hydrolysis 5’Phosphate + 3’Hydroxyl – Phosphate ester Transformation of E. coli Selection of transformed colonies (selectable marker on plasmid) Amplification of plasmid DNA in bacteria Screen for Recombinant Clones Insertional inactivation of gene on plasmid Antibiotic resistance (pBR322) Lac Z gene (pUC19) Analysis of Recombinant Clones Restriction mapping DNA sequencing
Frequency of any 6 bp DNA sequence = (1/4)6 = 1/4096 bps Any 4 bp sequence = (1/4)4 = 1/256 bps Any N bp sequence = (1/4)N
Standard Recombinant DNA technique: Cloning a DNA fragment into a plasmid (up to 10 kb DNA fragments) Insert = Donor DNA = Genomic DNA Vector = Plasmid = Engineered Bacterial Plasmids (pBR322, pUC19..) Restriction Enzymes = bind and cut at palindromic sequences Digest insert and vector Leaves Cohesive (sticky) ends = 5’ or 3’ overhang Leaves Blunt ends DNA Ligase = catalyzes phosphoester bond formation using energy of ATP hydrolysis 5’Phosphate + 3’Hydroxyl – Phosphate ester Transformation of E. coli Selection of transformed colonies (selectable marker on plasmid) Amplification of plasmid DNA in bacteria Screen for Recombinant Clones Insertional inactivation of gene on plasmid Antibiotic resistance (pBR322) Lac Z gene (pUC19) Analysis of Recombinant Clones Restriction mapping DNA sequencing
Standard Recombinant DNA technique: Cloning a DNA fragment into a plasmid (up to 10 kb DNA fragments) Insert = Donor DNA = Genomic DNA Vector = Plasmid = Engineered Bacterial Plasmids (pBR322, pUC19..) Restriction Enzymes = bind and cut at palindromic sequences Digest insert and vector Leaves Cohesive (sticky) ends = 5’ or 3’ overhang Leaves Blunt ends DNA Ligase = catalyzes phosphoester bond formation using energy of ATP hydrolysis 5’Phosphate + 3’Hydroxyl – Phosphate ester Transformation of E. coli Selection of transformed colonies (selectable marker on plasmid) Amplification of plasmid DNA in bacteria Screen for Recombinant Clones Insertional inactivation of gene on plasmid Antibiotic resistance (pBR322) Lac Z gene (pUC19) Analysis of Recombinant Clones Restriction mapping DNA sequencing
Standard Recombinant DNA technique: Cloning a DNA fragment into a plasmid (up to 10 kb DNA fragments) Insert = Donor DNA = Genomic DNA Vector = Plasmid = Engineered Bacterial Plasmids (pBR322, pUC19..) Restriction Enzymes = bind and cut at palindromic sequences Digest insert and vector Leaves Cohesive (sticky) ends = 5’ or 3’ overhang Leaves Blunt ends DNA Ligase = catalyzes phosphoester bond formation using energy of ATP hydrolysis 5’Phosphate + 3’Hydroxyl – Phosphate ester Transformation of E. coli Selection of transformed colonies (selectable marker on plasmid) Amplification of plasmid DNA in bacteria Screen for Recombinant Clones Insertional inactivation of gene on plasmid Antibiotic resistance (pBR322) Lac Z gene (pUC19) Analysis of Recombinant Clones Restriction mapping DNA sequencing
Standard Recombinant DNA technique: Cloning a DNA fragment into a plasmid (up to 10 kb DNA fragments) Insert = Donor DNA = Genomic DNA Vector = Plasmid = Engineered Bacterial Plasmids (pBR322, pUC19..) Restriction Enzymes = bind and cut at palindromic sequences Digest insert and vector Leaves Cohesive (sticky) ends = 5’ or 3’ overhang Leaves Blunt ends DNA Ligase = catalyzes phosphoester bond formation using energy of ATP hydrolysis 5’Phosphate + 3’Hydroxyl – Phosphate ester Transformation of E. coli Selection of transformed colonies (selectable marker on plasmid) Amplification of plasmid DNA in bacteria Screen for Recombinant Clones Insertional inactivation of gene on plasmid Antibiotic resistance (pBR322) Lac Z gene (pUC19) Analysis of Recombinant Clones Restriction mapping DNA sequencing
Add restriction enzyme recognition sites at the end of cDNA Cloning of cDNA fragments cDNA synthesis from mRNA cDNA cloning into plasmid vector, transformation of E. coli, …etc Add restriction enzyme recognition sites at the end of cDNA Clone by inserting into plasmid same as with genomic DNA
Cloning genomic DNA into Bacteriophage Lambda (~ 20 kb inserts; high frequency of introducing DNA into E. coli “transduction”; selects for recombinants directly) Vector = Lambda Phage DNA “arms” In vitro packaging = formation of virus particles in vitro using protein + recombinant phage clone Cloning Genomic DNA into Cosmid Vectors (~30-40 kb inserts; high frequency of introducing into E. coli, selects for recombinants) Vector = plasmid containing lambda COS site In vitro packaging, … etc
Cloning into Bacterial Artificial Chromosome Vector (BAC) (150-300 kb fragments; for isolation of large genes and analysis of genome structure) Vector = BAC vector = part of E. coli F1 phage Transformed into E. coli,… etc
Polymerase Chain Reaction PCR
Polymerase Chain Reaction PCR
Polymerase Chain Reaction PCR
Screening for Specific Genes in a Collection of Recombinant Clones Collection = Library = all possible DNA sequences present in at least one clone in the collection = “complete” library Genomic library cDNA library Vector depends on size of insert needed and size of library needed Method used to screen (search) for desired clone depends on information available Steps in the screen: Plate bacteria/virus on petri plates (colonies/plaques) Make replica of plates on filters: DNA from clones exposed and denatured on the filters “Probe” filter to identify specific clone Pick colony/plaque identified by probe, amplify in bacteria, analyze use as desired… Probes for specific genes: 32-P labeled DNA if DNA sequence of gene or amino acid sequence of protein is known oligonucleotides synthesized in vitro (commercially available homologous gene PCR product Antibody to protein product Vector must be an “expression vector” where the cloned gene is expressed in E. coli
Screening for Specific Genes in a Collection of Recombinant Clones Collection = Library = all possible DNA sequences present in at least one clone in the collection = “complete” library Genomic library cDNA library Vector depends on size of insert needed and size of library needed Method used to screen (search) for desired clone depends on information available Steps in the screen: Plate bacteria/virus on petri plates (colonies/plaques) Make replica of plates on filters: DNA from clones exposed and denatured on the filters “Probe” filter to identify specific clone Pick colony/plaque identified by probe, amplify in bacteria, analyze use as desired… Probes for specific genes: 32-P labeled DNA if DNA sequence of gene or amino acid sequence of protein is known oligonucleotides synthesized in vitro (commercially available homologous gene PCR product Antibody to protein product Vector must be an “expression vector” where the cloned gene is expressed in E. coli