Microbial biotechnology

• Define a clone in a cDNA library

  This is cDNA which has been inserted into a vector and replicated in a host cell.
• What are the steps in producing recombinant proteins

  Collect DNA.

  Clone gene into a vector.

  Transfer vector into host.

  Host expresses gene.

  Harvest protein.
• What is the benefit of clone libraries

  You just order the clone you want and the genome is already sequenced.
• What is a cDNA library

  This is a collection of cDNA clones that have been synthesized from mRNA.
• What type of libraries include non-coding regions

  Genomic
• What type of libraries only include exons

  cDNA
• What library is used to reconstitute a genome

  Genomic
• What libraries are used to reconstititute proteins

  cDNA
• What library is used to study gene expression

  Genomic
• What library is used to study gene function

  Genomic
• What library is used to study allele variation

  Genomic
• What library is used to produce proteins

  cDNA
• What library is used to study protein structure and/or activity

  cDNA
• Why do we clone cDNA copies into vectors and then a host

  It will contain only exons because bacteria are unable to splice out introns.

  mRNA is to unstable to clone directly.
• What are the steps in producing a cDNA library

  1. Isolate mRNA from a tissue sample
  2. Produce cDNA using reverse transcriptase and DNA polymerase
  3. Ligate into a plasmid using a restriction endonuclease and DNA ligase
  4. Transform into host to produce a clone
  5. Culture clones
  6. Screen the library
  7. Isolate and purify plasmids
• What is a DNA probe

  A labelled sequence of DNA which is used to detect DNA sequences.
• What are the methods to screen cDNA libraries

  Hybridization using labelled probes.

  Immunodetection using antibodies.

  Complementation using mutant deficiencies.
• Which methods of screening cDNA libraries depends on DNA sequence

  Hybridization
• Which methods of screening cDNA libraries depends on the properties fo the protein

  Immunodetection and complementation
• What is the aim of hybridization for a cDNA library

  To locate the cDNA clone using a DNA probe
• What are the types of hybridization probes for cDNA

  Homology and degenerate
• Describe homology probes

  The probe carries a complementary DNA sequence from a related organism.

  The probe will not be identical to the cDNA.
• Describe degenerate hybridization for cDNA

  The probe carries a degenerated sequence which is inferred from the protein sequence.
• Describe how degenerate oligonucleotides are used in cDNA screening

  You determine the possible DNA sequences form the protein sequence.

  choose the least degenerated sequence for your probe.

  The first method:

  Mix your least degenerated sequence with labelled nucleotides.

  Use the same label for each nucleotide.

  Only one possible combination of nucleotides will be a perfect match.

  Method 2:

  Use labelled nucleotides and inosine artificial nucleotides, these bind where multiple bases are possible.

  This allows you to probe to a larger sequence of DNA.
• What are the types of labelles used in cDNA hybridization

  Radioactive phosphorus and Didoxygenin.
• Describe the use of radioactive phosphurus as a probe label

  The nucleotide consists of 3 phosphate ions.

  The first ion is referred to as the alpha, and this is labelled with either phosphorus 32 or 33.
• What are the advantages of radioactive phosphurus as a probe label

  It is easy to detect at low levels.
• What are the disadvantages of radioactive phosphurus labels

  It has a short half-life.
• What are the advantages of digoxygenin as a probe label

  It can be frozen for long term storage.
• Describe how digoxygenin is used as a probe

  The oligonucleotide binds to the target.

  The digoxygenin is detected using an enzyme-linked antibody [Alkaline phosatase].

  This precipitates the DNA and produces chemiluminescence which is detected using photographic film.
• What are the two enzyme used to incorporate a label into DNA

  Terminal transferase and Taq polymerase
• Describe how taq polymerase is used to incorporate a label into DNA

  the dNTPs are labelled and incorporated during PCR. This incorporates the labels randomly.
• Describe how terminal transferase is used to incorporate labels into DNA

  Amplify the DNA probe using PCR.

  Then, add the labelled dNTPs to the 3 end.
• How is cDNA library screened using colony hybridization

  Use a filter to produce a DNA stamp of each colony.

  Denature the DNA and then anneal the probe.

  Use autoradiography to identify the colonies with the cDNA.
• What is autoradigraphy

  The detection of radiation using an X-ray.
• What are the steps in immunodetection of a cDNA library

  The cDNA clone produces the protein.

  Transfer the proteins to a nitrocellulose membrane which has a high affinity for proteins.

  Probe with a labelled primary antibodies that detect the protein.

  OR you can you can use an additional enzyme-labelled secondary antibody that binds to the primary antibody. This is labelled with alkaline phosphatase.
• How are protein specific antibodies made

  Immunize a mouse with the whole or partial protein.

  Isolate the antibody producing cells.

  Fuse with a myeloma to form a hybridoma.

  Screen the hybridomas for the correct antibody.

  Clonal expansion and isolate the monoclonal antibodies.
• How is a cDNA clone forced to express a protein

  Produce a phage lambda cDNA clone.

  The phage lambda infects E.coli.

  The E.coli replicates the lambda phage and produces the protein.

  When the E.coli dies the protein is released.
• What is complementation screening

  The detection of clones using the activity of the protein.
• Describe complementation screening for E.coli using the histidine synthesis enzyme 


  The mutants are unable to grow if histidine is present.

  1. Ligate the plasmid and cDNA containing histidine synthesis enzyme
  2. Transform into the mutants
  3. Culture in a liquid medium with histidine
  4. Culture on agar without histidine

  Only the mutants that took up the cDNA will grow.