Clone Human Gene from cDNA Libraries - Essay Example

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 a 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 of genetic science. Before delving deep into the techniques used to clone from cDNA libraries, it is important 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 a 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 DNA 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 labelled CDNA probe is not available. In this technique, it is the knowledge about a CDNA’

CDNA’s sequence is used to quantify an expression. Like other PCR-based screening methods, this technique is used to isolate novel cDNAs. In these methods, the moment a single or two members of a family are identified, other regions of homology are identified since they are always conserved within families (McLaren, 2000). In addition, PCR primers are designed, developed and used to amplify reverse transcriptase products of messenger RNA in the appropriate tissues.

Because cDNAs do not have specific ends (restriction sites or sticky ends), it is necessary that they are first manipulated for efficient joining to a vector DNA molecule in cDNA cloning. However, for blunt-ended cDNAs, direct cloning into a vector that has already been cut with a blunt end-cutting restriction enzyme (Yeku & Frohman, 2011). It is worth noting that blunt-end ligations are normally less efficient in comparison with sticky end ligations. Hence, it is normal to encounter cDNAs with cohesive ends.

A rather commonly used technique in the efficient cloning of cDNAs is via the production of cohesive ends on cDNAs and plasmid. This technique exploits the activity of the terminal transferase, a template-independent DNA polymerase. The function of this enzyme, produced by the calf thymus, is to catalyse the addition of 'homopolymer tails' to the 3' ends of the DNA. However, this addition only happens in the presence of a single base nucleotide such as dCTP (Yeku & Frohman, 2011). The terminal transferase thus builds up a string of C's (poly C tail) at the two ends of the DNAs.

The Polymerase Chain Reaction, which uses the thermo-stable DNA polymerase Taq polymerase, is also used in cloning human genes from cDNA. This technique is especially preferable in situations where only small amounts of messenger RNA are available.