Recombinant DNA Technology Prof. Elena A. Carrasquillo Chapter 4 Molecular Biotechnology Lecture 4

Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

Kinds of Libraries Genomic Library: Stores a representation of the genome (at least one copy of a gene present) plasmid, bacteriophage, phagemid, cosmid vectors cDNA Library: Stores a representation of the mRNAs expressed at a certain time or stage by a microorganism or organism plasmid, bacteriophage, phagemid, cosmid vectors

How is a Library Built: Restriction Enzyme Mechanisms: Preparation of DNAs to be joined (a)Staggered cut: leaves “sticky ends”

How is a Library Built: Restriction Enzyme Mechanisms: Preparation of DNAs to be joined: (b) Blunt End

Ligation of DNA cut with a Restriction Enzyme Staggered “sticky ends”

Ligation of DNA cut with a Restriction Enzyme Role of T4 DNA Ligase

Choosing the Vector Depends on the size of DNA to be cloned Is the protein encoded by the DNA going to be expressed in a prokariotic or eukaryotic cell?

Restriction Enzyme Map Allows ordering of DNA fragments

Restriction Enzyme Map We can then build maps of linear and circular molecules

Plasmid: it’s a circular DNA molecule containing: a multiple cloning site or MCS an antibiotic resistance gene an origin of replication pBR322 is the basis of most engineered plasmids

Selectable Markers:

Kinds of plasmids in the wild: F plasmids-transfer information from cell to cell R plasmids-confer antibiotic resistance Degradative plasmids-utilization of unusual metabolites Cryptic plasmids-No apparent function

Other characteristics: Size range form less than 1 kb to more than 500 kb Origin of replication-allows plasmid to replicate in the bacteria Low-copy number: 1-4 per cell High copy number: per cell Incompatibility groups: different kinds cannot be inside the same cell

Characteristics of an engineered plasmid: Small size (<15Kb) for optimal efficiency of transformation in bacteria Unique restriction enzime sites for cloning One or more selectable genetic markers to allow for differentiation of the plasmids carrying the cloned DNA vs the religated ones.

Plasmid: a cloning vector or vehicle

pUC19 another plasmid cloning vector Characteristics: Interruption of b-lactamase gene gives rise to white colonies (cloned DNA) vs blue ones (empty)

MCS: multiple cloning site of pUC19: -Produced by site-directed mutagenesis to alter the DNA sequence but not the protein sequence of b-lactamase -This produced new restriction enzyme sites

Creation of a DNA Library: Partial DNA digestion Purpose: To produce overlapping DNA fragments

Progress of Reaction: agarose gel electrophoresis Vary time of digestion or amount of enzyme units

Screening a library:I. Colony hybridization or Southern Blot

Preparation of DNA Probe Method 1 Random primers Enzyme: Klenow Fragment + dNTPs Non-radioactive: biotin or chemiluminescent labeled dNTPs -Radioactive: 32 P

Preparation of DNA Probe Method 2: 5’-end labeling Method 3: 3’end labeling

Klenow Fragment of E. coli DNA Polymerase I Enzyme Activities The polymerase (red) adds deoxyribonucleotides to the 3’- hydroxyl groups of the growing chains -The 5’ exonuclease (blue) removes succesive nucleotides from the 5’ phosphate ends -The 3’ exonuclease (yellow) removes succesive nucleotides from the3’ hydroxyl ends

Screening a Genomic Library Colony Hybridization

Colony Hybridization: Procedure:

Screening a Genomic Library: II.Colony Immunoassay The probe is an antibody The colonies are grown so that the recombinant protein is expressed

Screening a Genomic Library: II.Colony Immunoassay If chemiluminescense was used, light will be emitted and captured in an X-ray film

Screening a Genomic Library: III.Functional Complementation Defective host cells (A-) are transformed with a genomic library derived from cells that are normal with respect to that function (A+) and grown in minimal media. Those cells harboring a plasmid that corrects the defect will grow in minimal media.

Another Type of Library:cDNA Library Used to obtain functional eukaryotic coding regions. E. coli does not process introns. First step: Isolate poly A+ mRNA with oligo (dT) cellulose

cDNA Library: Second Step:Synthesis of cDNA from mRNA of specific cells

cDNA Library: Third Step:Selecting and Cloning Full length cDNA molecules

Genomic Library: Bacteriophage λ (Lambda) For cloning inserts of Kb Plasmid libraries hold up to 10 kb inserts

Genomic Library: Bacteriophage λ (Lambda)Life Cycle Lytic Cycle:Production of progeny Lysogenic Cycle: Integration into bacterial chromosome

Genomic Library: Bacteriophage λ

Cosmid library Allows cloning of 45 kb DNA fragments

BACs: Bacterial Artificial Chromosomes Based on P1 bacteriophage, the F plasmid and the lacZ region of pUC plasmids It’s a low copy number plasmid Carries kb fragments

BACs: Bacterial Artificial Chromosomes

YACs:Yeast Artificial Chromosomes

Methods of Introducing Foreign DNA Transformation Electroporation Conjugation

Electroporation Application of an electrical field to cells

Conjugation:Tripartite mating