Illumina method of DNA sequencing analysis
The Illumina method of DNA sequencing has been extensively used in biochemistry, the results of the positive target sites in the different strain of DNA have been identifying and it possesses high speed or has the rapid capacity to do the sequencing efficiently. This illumine technique is based on the cyclic reversible termination. This method can be used for methylation profiling, small RNA discovery, and transcriptome analysis. The three important steps for this method are amplifying, sequence, and analyze. In this method, a flow cell is required, in which the DNA is loaded to get the multiple copies at the end of this mechanism. Adapters are also required that will work as the reference points at the time of all the three stages of this technique. Nano walls are present in the flow cell. Every nano-well is well equipped with the oligonucleotides, these assist in the binding with the adaptor to remain bounded or attached. The cloning of the DNA is carried by the polymerase chain reaction.
The genomic library is prepared and the DNA is purified. The genetic material is cut into the different fragments into the sizes between fifty to five hundred base pair fragments. In these fragments of genetic material, adaptors are added. The sonification process is carried by using ultrasonic sound waves. The adapters used in this process include different segments, these sequences have complementary bases and these are needed for binding of the sequencing primer. Acrylamide coating is done in the glass flow cell; the flow of this technique has short nucleotide sequences. Whenever the segmented genetic material is washed present on the flow cell, the useful adapters binds with the complementary bases.
Bridge amplification:
As soon as the cluster formation initiates, this leads to the formation of several same strands of DNA. In these strands, a few of them may be forward and the remaining strands may be of reverse. Due to this forward and reverse strand, the left and right adapters are required for the bridge formation. The enzyme DNA polymerase helps in polymerizing the DNA strands. Thus it helps in the formation of the complementary strands. Adaptor sequence is also present at the top region of the reverse strand of DNA. The bending of the DNA strand occurs and it assists in the formation of the complementary strands.
Amplification of the cloned DNA:
The DNA segment bends and it gets fixed with some solid support. The double-stranded DNA is polymerized with enzyme DNA polymerase. New strands of DNA are generated. The clonal amplification is done and is vital for quality control. The reverse strands of the genetic material get washed from the flow cell and in the flow cell, only forward strands are found at that time. The polymerase enzymes also help in the addition of the fluorescently tagged deoxynucleotide triphosphate (dNTP) in the DNA strand. To activate the fluorescent-labeled bases, the laser is passed on the flow cells and this kind of fluorescence is identified with the help of a camera, and the data is managed through the computer. Each of the bases gives a different type of color. The sequences of the genetic material are analyzed base on the base at the time of the Illumina sequencing technique. At the end of the reaction, the 3’ end of the genetic material is blocked. The washing is done in the forward strand. This method of sequencing is useful to arrange the different genome sequences and this makes the comparison or the differentiation among the DNA sequences to improve the different health issues of humans.
