Availability of sufficient quantities of sufficiently pure protein for production of specfic antibodies or for partial peptide sequencing opens the doors to cloning of the gene specifying that protein.
- Physiological and biochemical investigations may lead to the identification of a protein responsible for the phenotype or biological property of interest.
- If the purified polypeptide has a non-blocked N-terminus, the N-terminal sequence may be determined directly.
- Sequences from the interior of the protein can be obtained by producing and analyzing proteolytic fragments of the polypeptide.
- If sufficient quantities of purified protein are available, the protein may be used to immunize animals (commonly rabbits or mice). The animals usually produce, and secrete into the serum, antibodies specifically recognizing the protein. The antisera may be used to detect the immunizing protein specifically. Top
- Peptides of 10 to 30 residues in length can be chemically synthesized efficiently. Top
- Small peptides chemically coupled to larger carriers can be effective immunogens. Top
- Nucleotide sequences that could code for the determined sequence of amino acids can be deduced from the genetic code. Since the genetic code is redundant, multiple nucleotide sequences can encode the same peptide sequence. To be sure that the actual nucleotide sequence is present in a probe oligonucleotide, the oligonucleotide is synthesized incorporating, where needed, multiple nucleotides. The product is called a degenerate oligonucleotide. Top
- Antisera can be used to recognize clones of an expression library that are synthesizing the cognate antigen. The antibodies bind to protein from the colony or plaque. Bound antibodies can be detected by any of a variety of methods. Top
- When amino acid sequences from two separated parts of the polypeptide chain are available, degenerate oligonucleotides based on the two sequences can be used to attempt to amplify the intervening sequence by PCR. Top
- An amplified RT-PCR product or a degenerate oligonucleotide may be used in nucleic acid hybridization to screen the colonies or plaques of a cDNA library for clones containing complementary sequences. Top
- Positive clones obtained from an ordinary or an expression cDNA library can be used as nucleic acid hybridization probes to screen a library of genomic DNA to identify clones containing the gene that can produce the corresponding mRNA. Top
- Positively reacting genomic library clones need to be further examined to :
- identify where on the clone the gene is located; and
- prove that the clone actually contains the gene.
- A sterling example of the use of protein-based cloning is the identification of 44 (possibly all) polypeptides that are components of the spliceosome (ref).
- Polypeptides were separated from highly purified spliceosomes.
- Fragments of each polypeptide were generated by enzyme digestion.
- The sequences of selected fragments were determined by nanoelectrospray mass spectroscopy.
- The fragmentary sequences were used to identify nucleotide sequences encoding them by a search of a database of known cDNA sequences (an EST database).
- These nucleotide sequences were used to obtain complete cDNA clones for each polypeptide.
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This is page 415 of Molecular Genetics by Ulrich Melcher, ©1997, 1998, 1999
E-mail inquiries to U. Melcher------------Last Updated: 14 October, 1999