In higher eukaryotes, an mRNA is a more useful predictor of a polypeptide sequence than is a genomic sequence, because the introns have been spliced out. The cDNA is made from mRNA with the use of a special enzyme called reverse transcriptase, originally isolated from retroviruses.
Using an mRNA molecule as a template, reverse transcriptase synthesizes a single-stranded DNA molecule that can then be used as a template for double-stranded DNA synthesis. Clear All. Products Per Page: 8 12 16 20 40 Columns: 1 2 3 4 6. A cDNA library represents a sampling of the transcribed genes, but a genomic library includes untranscribed regions.
First of all, it involves the isolation of total mRNA from a cell type or tissue of interest. It may be desirable to remove highly abundant tRNAs and rRNAs which might otherwise constitute the majority of the final library to the detriment of the detection of low abundance RNAs.
It is first converted into DNA prior to insertion into a suitable vector. It is essential to use only the minimal number of amplification cycles needed to obtain sufficient material for sequencing to avoid over-amplification of the libraries, which is a major source of bias in the results.
Because the blunt-end ligation is inefficient, short restriction-site linkers are first ligated to both ends. To isolate and purify RNA, a variety of strategies are available depending on the type of source materials e. The main goals of isolation workflows are to stabilize RNA molecules, to inhibit RNases, and to maximize yield with proper storage and extraction methods. Optimal purification methods remove endogenous compounds, like complex polysaccharides and humic acid from plant tissues that interfere with enzyme activity; and common inhibitors of reverse transcriptases, such as salts, metal ions, ethanol, and phenol.
Contaminating gDNA can interfere with reverse transcription and may lead to false positives, higher background, or lower detection in sensitive applications such as RT-qPCR. Their thermolabile property allows simple inactivation at a relatively mild temperature e. Most reverse transcriptases used in molecular biology are derived from the pol gene of avian myeloblastosis virus AMV or Moloney murine leukemia virus MMLV.
The MMLV reverse transcriptase became a popular alternative due to its monomeric structure, which allowed for simpler cloning and modifications to the recombinant enzyme. These attributes result in increased cDNA length and yield, higher sensitivity, improved resistance to inhibitors, and faster reaction times Table 1. Maintaining RNA integrity is critical and requires special precautions during extraction, processing, storage, and experimental use see step 1 :.
RNase inhibitor. Often included in the reaction buffer or added to the reverse transcription reaction to prevent RNA degradation. They may be:. A number of known RNases exist, and appropriate RNase inhibitors should be chosen based on their mode of actions and reaction requirements.
Contaminating RNases cannot be removed by simple filtration, and autoclaved water is not adequate because RNases are heat stable. Reverse transcription reactions involve three main steps : primer annealing, DNA polymerization, and enzyme deactivation. The temperature and duration of these steps vary by primer choice, target RNA, and reverse transcriptase used. The critical step is during DNA polymerization.
In this step, reaction temperature and duration may vary according to the primer choice and reverse transcriptase used. Among reverse transcriptases there are differences in thermostability, which in turn determines the highest optimal polymerization temperature for each.
Using a thermostable reverse transcriptase allows, a higher reaction temperature e. With such enzymes, high-temperature incubation can result in an increase in cDNA yield, length, and representation. In contrast, an engineered reverse transcriptase with high processivity may take as little as 10 min to synthesize a 9 kb cDNA. Watch the webinar. Don't have an account? Create Account.
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