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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
sequencers
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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 Scheme
Make library
Plate library
Denature and transfer
DNA to membranes
Make labeled probe
Dry and autoradiograph
or detect
Wash
Denature &
Hybridize
Library Construction
• Genomic l Library
– Isolate Genomic DNA
– Limited Digest with
restriction enzyme
– Analyze digest with gel
electrophoresis
– Prepare l arms
– Ligate digest to arms
– In vitro package l
– 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
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DNA template preparation
Nick template with DNase I
Fill in gaps with DNA polymerase and labeled nucleotides
Denature and hybridize
• Random Priming
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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
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Clone template into a T7, T3 or Sp6 vector
Restriction cut to linearize
RNA polymerization with labeled rNTPs
Denature and hybridize
Labels
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Radioisotopes
Fluorescent
Colorimetric
Antibody
Random Priming
End Labeling
Digoxygenin Labeling
Hybridization Stringency
Clone Characterization
Characterization Scheme
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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
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PCR and Cloning
Protein Expressing Libraries
Overall Scheme
Isolate RNA
Make cDNA
Ligate into expression
Vector and transform/
transduce
Plate and transfer
protein to membrane
Detect with colorimetric
or fluorescent technique
Wash
Incubate with
1º and 2º
antibodies