Synthesis of cDNA and cDNA library

Introduction

cDNA is called complementary DNA. The cDNA synthesis takes place from a mRNA (messenger RNA) or miRNA (micro RNA). This transcription process (Synthesis) takes place with the help of the enzyme reverse transcriptase.

The basic difference between cDNA and genomic DNA is that they lack introns or non-coding sequences. Genomic DNA is composed of both introns (non-coding) and exons (coding sequences). Introns are mostly present in higher eukaryotes and rarely present in bacteria. Due to the absence of introns, the size of cDNA is significantly smaller than the genomic DNA.

Definition of cDNA and cDNA library

cDNA is the copy or complementary DNA produced by using mRNA as a template by using reverse transcriptase enzyme.

cDNA library

A cDNA library is a collection of cloned DNA sequences that are complementary to those extracted from the cell or tissue.

What is a cDNA library?

• cDNA library is a combination of cloned cDNA fragments inserted into host cells and stored as a “library”.
• It is produced from fully transcribed mRNA which contains only the expressed genes of an organism.
• cDNA library is prepared by reverse transcribing a population of mRNAs and then screened for those respective clones.
• The cDNA library is representative of the RNA population from which it was driven.
• It can be enriched for most of the mRNAs but may contain only some of the clones representing rare mRNAs.
• An appropriate strategy to obtain cDNA in abundant quantity is to clone them in an M13 vector. For the separation of cDNA clones in moderate and low abundance classes, it is necessary to construct a cDNA library.
• Generally, Phage lambda insertion vectors are well suited for cDNA cloning and some of them are most widely used like λgt10 and λgt11 (1).

cDNA preparation

• The creation of a cDNA library begins with messenger ribonucleic RNA instead of DNA.
• mRNA carries encoded data from DNA to ribosomes for translation.
• cDNA library can be prepared by isolating mRNA from tissue, which is actively synthesizing proteins like roots, leaves, etc.
• The mRNA is used for replicating it into cDNA through the process of reverse transcriptase.
• cDNA molecule can be made double-stranded and cloned. Will differ from genomic libraries as it is lacking the intron, but may have the advantage expressed in bacteria.

Synthesis of cDNA

The synthesis of cDNA takes place by using the following steps.

• A cDNA library is prepared by isolating mRNA from a particular population of cells. mRNA isolated from eukaryotic cells has the poly-A tail (a string of adenines) on the 3’end.
• The next step is to mix mRNAs with oligo(dT) primers short, single-stranded sequences of T nucleotides that anneal to the poly-A tail.
• The enzyme reverse transcriptase extends the oligo (dT) primer and synthesizes a complementary DNA copy of the mRNA sequence.
• From this reaction, an mRNA-DNA double-stranded hybrid is formed. The RNA in the hybrid molecule can be chemically degraded or enzymatically digested.
• After this, the opposing strand of DNA is synthesized by using DNA polymerase.
• The cDNA molecules are then subsequently inserted into vectors (usually plasmids).
• To insert cDNA into a plasmid linker sequence should attach to the ends of the cDNA.
• Linkers are short double-stranded oligonucleotides containing a restriction enzyme recognition sequence (e.g., EcoRI).
• After attachment to the cDNAs, the linkers are cut with EcoRI and ligated to vectors treated with the same enzyme.
• Transfer of vectors carrying cDNA molecules to host cells and cloning is used to make a cDNA library.
• cDNA libraries give certain advantages over genomic libraries and are a useful method for gene cloning.
• This is due to a cDNA library containing DNA copies called cDNA which is made from the mRNA molecules of a cell population.
• cDNA is complementary to the nucleotide sequence of the mRNA. Whereas a genomic library contains all the DNA in a genome (gene coding and noncoding sequences), a cDNA library contains only expressed genes.
• As a result, cDNA libraries have been particularly useful for studying and identifying genes expressed in certain cells or tissues.

Uses of cDNA library

• Synthesis of cDNA is used to study gene expression via methods such as RNA sequencing or RT-PCR.
• It determines which mRNAs are expressed at different developmental stages.
• cDNA library is used for the isolation of the gene that codes for that corresponding mRNA.
• To study the alternative splicing in different cells.
• cDNA libraries are used to discover novel genes.
• These libraries provide information on the genes that were transcriptionally active in a tissue at a particular time.
• Many different cDNA libraries are available from cells and tissues in specific stages of development.
• These libraries provide an instant record of all the genes active in a cell at a specific time.
• It is a very valuable tool for scientists to isolate and study genes in particular cells or tissues.

Q&A

1. What are histones?

Histones are the proteins that are associated with chromosomes. About 80% of chromosomal proteins are histones. They are a highly heterogeneous class of proteins.  The molecular weight of histones generally ranges between 10000 to 30000. Tryptophan is absent in histone protein. Histone protein constitute of H1 (rich in lysine), H2a (slightly rich in lysine), H2b (slightly rich in lysine), H3 (arginine-rich), and H4 (arginine-rich). Histones play a primary function in the chromosomal organization.

 2. Why do histones bind tightly to DNA?

One micron of a metaphase chromosome contains about 8000 microns of DNA double helix. The packaging of such a long DNA molecule into a small segment of the metaphase chromosome is difficult. So, to arrange the maximum DNA into small segments, histone protein works. Thus it can be said that the Histone is the protein of the DNA, which binds the DNA tightly to make it smaller. Scientists discovered that Histone fractions like H2a, H2b, H3, and H4 are involved in the structural organization of chromatin fibers and H1 holds the folded chromatin fibers of chromosomes.

Written By: Pallavi Bhoyar