Molecular Tools

Recombinant DNA Restriction enzymes Vectors Ligase and other enzymes

DNA Cloning Restriction enzymes Plasmid vector –Selectable gene –Cloning sites –Origin of replication DNA ligase Transformation –Competent cells Selection

Dideoxy DNA Sequencing Dideoxy nucleotides Automated DNA sequencers

Polymerase Chain Reaction

Thermostable DNA polymerase Oligonucleotide primers NTPs + buffer PCR machine

Constructing, Screening & Selecting Recombinant Clones

Overall Scheme Make libraryPlate library Denature and transfer DNA to membranes Make labeled probe Denature & Hybridize Wash Dry and autoradiograph or detect

Library Construction Genomic  Library –Isolate Genomic DNA –Limited Digest with restriction enzyme –Analyze digest with gel electrophoresis –Prepare arms –Ligate digest to arms –In vitro package –Infect and plate cDNA/expression –Isolate RNA –Poly A isolation via oligo dT column –Reverse transcribe mRNA –Digest or hydrolyze mRNA –2nd strand synthesis –Add linkers –Prepare vector with digest and phosphatase –Ligate, transform and plate

cDNA Library

Genomic Library

Probe synthesis Nick Translation –DNA template preparation –Nick template with DNase I –Fill in gaps with DNA polymerase and labeled nucleotides –Denature and hybridize Random Priming –DNA template preparation –Anneal with random hexamers –Primer extend with DNA polymerase and labeled nucleotides –Denature and hybridize

Probe synthesis End labeling –Prepare template –End label with labeled ATP and polynucleotide kinase –Denature and hybridize RNA probes –Clone template into a T7, T3 or Sp6 vector –Restriction cut to linearize –RNA polymerization with labeled rNTPs –Denature and hybridize

Labels Radioisotopes Fluorescent Colorimetric Antibody

Random Priming

End Labeling

Digoxygenin Labeling

Hybridization Stringency

Clone Characterization

Characterization Scheme Grow up the isolated clones Make DNA from those clones Restriction digest characterization Blot to membrane and hybridize with labeled cDNA to map transcript Auto radiograph Subclone and DNA sequence

DNA minipreps

PCR and Cloning

Protein Expressing Libraries

Overall Scheme Isolate RNAMake cDNA Ligate into expression Vector and transform/ transduce Plate and transfer protein to membrane Incubate with 1º and 2º antibodies Wash Detect with colorimetric or fluorescent technique