16 Cloning Strategies: An Overview
259
based on sequence similarity, the following should be considered, especially
when the sequence is derived from amino acid sequence.
Choosing the region to derive a sequence from
• A portion of the amino acid sequence should be chosen in which there are
amino acids that have only a few codons (e. g. Trp (one codon) rather
than Ser (six codons)).
• A region should be selected, which is likely to have been conserved during evolution and which is likely not to be interrupted by an intron (if
cDNA is tobe used in a PCR).
Choosing codons for amino acids
• All possible codons for each amino acid should be written down below
the amino acid sequence, and their relative codon usage in the target
species (as published in codon preference tables (Pesole, Attimonelli,
Liuni 1988) should be indicated behind each codon. An oligonucleotide
mixture can be designed that includes only the codons most frequently
used in the target species. The number of different molecules in the mixture is the product of the number of choices in each position in the sequence. Ideally, this number should be less than 256, although probes
with higher degeneracy have been used successfully for cloning by hybridization as well as by PCR.
• Inosine, which base pairs with adenine, cytosine and uracil can be subsituted for the nucleotide in the third codon position if this nucleotide is
T, Gor A.
Expression cloning
If the gene product in question can be identified by a functional assay, and if
there exists a eukaryotic cellline that is deficient in this function, then stable
or transient transfection with, e.g. human genomic DNA can be performed.
Transfeeted cells can be assayed for reconstitution of the desired function.
Human DNA fragments retained in the cellline expressing the reconstituted
function can then be identified and the respective gene can be isolated, e.g.
by screening of a genomic library prepared from the transfected cellline.
259
based on sequence similarity, the following should be considered, especially
when the sequence is derived from amino acid sequence.
Choosing the region to derive a sequence from
• A portion of the amino acid sequence should be chosen in which there are
amino acids that have only a few codons (e. g. Trp (one codon) rather
than Ser (six codons)).
• A region should be selected, which is likely to have been conserved during evolution and which is likely not to be interrupted by an intron (if
cDNA is tobe used in a PCR).
Choosing codons for amino acids
• All possible codons for each amino acid should be written down below
the amino acid sequence, and their relative codon usage in the target
species (as published in codon preference tables (Pesole, Attimonelli,
Liuni 1988) should be indicated behind each codon. An oligonucleotide
mixture can be designed that includes only the codons most frequently
used in the target species. The number of different molecules in the mixture is the product of the number of choices in each position in the sequence. Ideally, this number should be less than 256, although probes
with higher degeneracy have been used successfully for cloning by hybridization as well as by PCR.
• Inosine, which base pairs with adenine, cytosine and uracil can be subsituted for the nucleotide in the third codon position if this nucleotide is
T, Gor A.
Expression cloning
If the gene product in question can be identified by a functional assay, and if
there exists a eukaryotic cellline that is deficient in this function, then stable
or transient transfection with, e.g. human genomic DNA can be performed.
Transfeeted cells can be assayed for reconstitution of the desired function.
Human DNA fragments retained in the cellline expressing the reconstituted
function can then be identified and the respective gene can be isolated, e.g.
by screening of a genomic library prepared from the transfected cellline.