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

QuickTime™ and aSorenson Video decompressorare needed to see this picture.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Dideoxy DNA Sequencing

• Dideoxy nucleotides• Automated DNA

sequencersQuickTime™ and a

Animation decompressorare needed to see this picture.

Polymerase Chain Reaction

Polymerase Chain Reaction

• Thermostable DNA polymerase

• Oligonucleotide primers

• NTPs + buffer• PCR machine

QuickTime™ and aSorenson Video decompressorare needed to see this picture.

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

QuickTime™ and aNone decompressor

are needed to see this picture.

DNA minipreps

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.QuickTime™ and a

TIFF (Uncompressed) decompressorare needed to see this picture.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

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