1. Material for Quiz 5: Chapter 8

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

9. 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

10. 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

11. 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

12. Cloning into Bacterial Artificial Chromosome Vector (BAC) ( 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

13. Polymerase Chain Reaction PCR

14. Polymerase Chain Reaction PCR

15. Polymerase Chain Reaction PCR

16. 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

17. 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