Chapter 7: Finding the Right Clone

Probes can be designed to bind specific or group of sequences with a region in common, method to label probe depends; type of template available, whether strand specific probe is needed, whether very sensitive probe is needed.

Screening Libraries with Gene Probes: Hybridization

  • Libraries must be screened to find cone carrying fragment of interest, use a modified DNA/RNA fragment or an antibody, strategy depends on what you know about target sequence
  • Bound probe must be detectable & quantifiable, radioactive/epitope tags (antibodies recognize epitopes)
  • Place a filter over colonies to pick up bacteria, peel the filter off and ensure DNA is attached to membrane, lay membrane on top of buffer to denature DNA and bake onto membrane, block filter with non-specific DNA or protein & incubate with probe solution
  • Radioactive probes detected via autoradiography (activates silver grains, dark spot film)
  • Denature target, fix to membrane, block, incubate with probe, wash off unbound, detect
  • Homologous Probe: Complementary to target (have part of sequence want the rest), parts of DNA
  • Heterologous Probe: Not exactly complementary, can detect gene family (forms of developing protein)
  • Distinct: Promoter, intron, UTR (not conserved, no selection pressure)
  • Hybridization Stringency: Measure of how restrictive the conditions are, high temp low salt increases
  • High Salt: Backbone charges are neutralized so even sequences that are not perfect will stay

Labelling Probes

  • Radiolabelled: 32P labelled probes can be detected by exposure to photographic film, usually incorporated in vitro DNA synthesis, a modified nt must be in alpha because beta and gamma hydrolyzed so not in DNA
  • Non-Radiolabelled: Modified nt can be detected with antibodies specific for the epitope tag
  • (DIG modified dUTP) = Fluorescence reaction

Methods to Incorporate Modified Nucleotides

  • Random Priming: Requires purified fragment from PCR, denatured fragment mixed with random hexamers (6nt), and Klenow polymerase (only incorporates nt no exonuclease activity), both new strands labelled, primers bind every 250nt on each strand so no good for short sequences
  • Labelled during DNA synthesis by PCR: Don’t need purified product (done on gDNA), dNTP in PCR
  • Labelling with T4 polynucleotide kinase: Removes ATP gamma P and adds to 5’ end, 1 per fragment
  • RNA probes: Insert cloned downstream of promoter, linearize plasmid by digestion at 5’ end, synthesize antisense transcript using RNA pol to recognize promoter, one of the ribonucleotides labels generating antisense riboprobe, most sensitive

Screening Libraries with Antibodies

  • If cDNA library is in expression vector, each transformant will express protein corresponding to cDNA so we can use an antibody specific to the protein encoded
  • Colonies spread onto plate, proteins transferred to nitrocellulose membrane not denatured, membrane incubated with primary antibody for protein, secondary antibody conjugated to detectable tag (radioactive, fluorescence, enzyme linked (add substrate and monitor rxn) binds to primary abundance of signal based on abundance in transcript, multiple 2ndary bind to one primary

Preparation of Antibodies

  • Inject purified protein into rabbit/chicken & isolate serum from blood, make based on amino acid sequence
  • Antigen can be a purified protein, or protein fragment, or synthesized polypeptide of 12-20 aa

Limitations

  • Need specific antibody and need good expression library with full length cDNA transcripts,
  • Expressed in non-native host may not fold correctly, antibody may be recognizing post-translational modification, may denature so antibody must recognize linear epitope

Cloning by Functional Complementation

It is possible to clone by complementation of a mutant phenotype in vivo, verifies the clone has been identified by another method and encodes the proposed activity in vivo, create a library and use it to transform a mutant: depending on which clone the mutant takes up it may allow it to survive a selective condition mostly done with cDNA/expression library to look for mutant who is now wildtype because they took up clone that fixed them

  • Use functional complementation to test if translation fusion protein has same functionality as protein alone to prove construct is not interfering with native protein function
  • Control
  • Mutant lack gene X, transform with construct ORF-X but no GFP & expect wildtype phenotype
  • Test
  • Mutant lack gene, transform with construct ORF-X-GFP and see if result matches

Limitations

  • Only works with transformable organisms, need visible phenotype, need high transformation efficiency

Southern Blotting

To know if there is a DNA fragment in your mixed DNA that can bind your probe, cannot retrieve colony

  • Test stringency of probe (whether cloned fragment binds a probe), Test whether clone has a fragment
  • Test whether genome contains sequence similar to probe, test number of gene family members in genome
  • Test how many copies of transgene in genome: PCR detects if transgene present, Southern blot quantifies
  • Restriction mapping of complex DNA: Can do restriction map using a probe

Steps

  • Digest DNA with RE and perform agarose gel electrophoresis with ethidium bromide and view fragments
  • Soak gel in NaOH to neutralize and denature DNA for hybridization
  • Filter paper immerse in high salt buffer with gel sitting on top of it, membrane with paper towels and weight above that and the buffer is drawn up by capillary action
  • Transfer the blot (gel) to nitrocellulose or nylon membrane using Southern blot technique
  • DNA fragments are fixed on membrane and now hybridize membrane with probe (no antibody)
  • Expose membrane to film (Membrane can be washed off and rehybridized under different stringency)

Controls

  • +ve: Electrophorese a dilution of the unlabeled fragment used to make the probe itself = band
  • Fragment diluted to same concentration as test target sequences to know if probe is sensitive and competes to bind to test samples, won’t see on ethidium bromide but will on auto radiography
  • RE not working, not enough NaOH so no ssDNA, wrong stringency, weight uneven
  • -ve: No DNA or sample should not bind probe, test contamination or probe dimers

Amount of Target Sequence Present: Digested DNA loaded * (target sequence length/genomic sample length)

Results: 2 lanes does not mean 2 copies of the gene, it could be that RE cut the one gene fragment

Benefits: Analyze multiple samples at once, can increase stringency slowly (not with DIG because reaction occurs with enzyme and you cannot remove that), modest equipment and cost

Limitations: Need probe so target sequence needed from same or similar organism, expensive for more sensitive, cant quantify only relative, can estimate gene copy / but RE can make it look like additional copies

Southern Blot vs Genomic Cloning

  • Southern Blot allows to determine if specific fragment is present and whether it has been cut (multiple digests to determine which one gives the gene in one piece) Southern blot tells us how many possible sequences in library and how long to screen for, helps design library by choosing RE and optimal stringency to make a library to actually isolate rather than just detect it, Southern blotting takes few days
  • Genomic cloning, we can use the RE that kept our gene in one piece


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