Wednesday, September 25, 2019

Gene cloning (Clone human gene from cDNA libraries...) Essay

Gene cloning (Clone human gene from cDNA libraries...) - Essay Example First, cloning is the separation or isolation of genetically homogenous strain of an organism (Lassen et al., 2005). Organisms at the same genetic level are identical within a clone. In most cases, bacteria, phages and even higher plants are cloned by the isolation of a single cell from the organism of interest and allowing the isolated cell to form a colony or an entire plant. In more specific terms, cDNA cloning entails the isolation of single but self-replicating organism followed by an amplification of its cell (Kfoury, 2007). However, there are certain conditions that should be met for such a technique to be treated as cDNA cloning. That is, such an organism’s DNA must contain the target cDNA. In cases where the interest of a researcher is in any cDNA produced by a given organism, it would not matter the type of cDNA produced. That is, any cDNA would work. The key and most difficult issue in many cloning studies on CDNA is never the isolation of CDNA but the selection of the CDNA of interest among many cDNAs. A DNA library on the other hand refers to a collection of various sequences of DNA combined into one vector (Kfoury, 2007). Thus, a CDNA library has sequences that are complementary to messenger RNAs. A vector refers to an organism that is designed for experimental purposes and self-replicates. In many experiments, vectors are made from bacteriophages, plasmids, retroviruses and animal viruses (Lassen et al., 2005). Vectors must have a system by which they reproduce, which is the essence genetic science. Before delving deep into the techniques used to clone from CDNA libraries, it is importance to overview a few cloning strategies. Cloning Strategies and Methods First among the recommended cloning strategies is the necessity of acquiring a library that contains the required sequence. Second, the clones of interest should be isolated. Third, formal tests to help ensure that the correct clones of interest have been isolated need to be developed. One method of creating human genes from CDNA is referred to as the Rapid Amplification of cDNA ends (RACE) (McLaren, 2000). The known strengths of RACE are that it is inexpensive and a powerful tool for acquiring full-range CDNA, even for partially known sequences. In this technique, which begins with a mixture of mRNAs, non-specification anchors and gene-specific primers generated from the known regions of the gene, it is possible to identify substitute transcripts of a gene for partial as well as complete sequence of only one known transcript (McLaren, 2000). This technique is used to obtain a full-range sequence of a cell’s RNA transcript. In the first step of the RACE process, reverse transcription in which an unknown end section of a transcript is copied by use of a known sequence from the middle of the transcript results in a CDNA copy of the RNA transcript. The copied region is bounded by the known sequence and either the 5' or 3' end. Screening of cDNA Libraries and D NA Synthesis of CDNA Inserts The other techniques by which human genes may be obtained from CDNA are screening of CDNA libraries and DNA synthesis of CDNA inserts. Screening of cDNA libraries, by transcript-specific RT-PCR cloning is particularly appropriate in situations where a labeled CDNA probe is not available. In this technique, it is the knowledge about a CDNA’

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