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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
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Dideoxy DNA Sequencing
• Dideoxy nucleotides• Automated DNA
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Polymerase Chain Reaction
Polymerase Chain Reaction
• Thermostable DNA polymerase
• Oligonucleotide primers
• NTPs + buffer• PCR machine
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Constructing, Screening & Selecting Recombinant Clones
Overall SchemeMake library Plate library Denature and transfer
DNA to membranes
Make labeled probe
Denature &Hybridize
WashDry and autoradiographor 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
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DNA minipreps
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PCR and Cloning
Protein Expressing Libraries
Overall SchemeIsolate RNA Make cDNA Ligate into expression
Vector and transform/transduce
Plate and transferprotein to membrane
Incubate with 1º and 2º antibodies
WashDetect with colorimetricor fluorescent technique