Dna

DNA ligase

| .and the nicks in the sugar-phosphate backbone are sealed by DNA ligase.

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18.14 A cDNA library contains only those DNA sequences that are transcribed into mRNA.

How is a probe obtained when the gene has not yet been isolated? One option is to use a similar gene from another organism as the probe. For example, if we wanted to screen a human genomic library for the growth-hormone gene and the gene had already been isolated from rats, we could use a purified rat gene sequence as the probe to find the human gene for growth hormone. Successful hybridization does not require perfect complementarity between the probe and the target sequence; so a related sequence can often be used as a probe. The temperature and salt concentration of the hybridization reaction can be adjusted to regulate the degree of complementarity required for pairing to take place. Alternatively, synthetic probes can be created if the protein pro duced by the gene has been isolated and its amino acid sequence has been determined. With the use of the genetic code and the amino acid sequence of the protein, possible nucleotide sequences of a small region of the gene can be deduced. Although only one sequence in the gene encodes a particular protein, the presence of synonymous codons means that the same protein could be produced by several different DNA sequences, and it is impossible to know which is correct. To overcome this problem, a mixture of all the possible DNA sequences is used as a probe. To minimize the number of sequences required in the mixture, a region of the protein is selected with relatively little degeneracy in its codons (I Figure 18.16).

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