Question

Please answer all questions please

1.Starting with normal and cancer intact cells as well as the preparation of the DNA microarray chip, explain in detail how the typical DNA microarray experiment will be performed in order to identify differentially expressed genes. What are the types of probes that can be used for this experiment, and how are they different? Explain how the data will be interpreted.

2.In a nutshell, describe the major advantages and disadvantages of each DNA sequencing platform

Answer 1

DNA microarray is used to detect more than one gene expression of a cell simultaneously. To execute a DNA microarray of a cancer cell as well as normal cell, steps will be followed as-

a) Isolation of mRNA - mRNA will be isolated from normal and cancer cell, one sample generally taken as control, and other sample taken as an experiment

b) Reverse transcription and labelling: To find the transcripts through hybridization, they required to be labeled, and due tolimited initial material there would be the use of an amplification step. Labeling generally includes a reaction of reverse transcription (RT) to generate a complementary DNA strand (cDNA) and inserting a florescent dye assosciated to a DNA nucleotide, generating a fluorescent strand of cDNA. Cancerous and Normal samples would be labeled with various dyes and combinely hybridized onto the same microarray in the succeeding step. Expressed genes reflect as fluorescent spots on immobilized genes.

c) Labeled target hybridized to the microarray. In this step labeled cDNAs positioned onto a DNA microarray where it can hybridize to their complementary probes of DNA fixed on the microarray. Unbound sequences removed by a series of washes. Microarrays gives a visual impression of gene expression. Then microarray would be scanned and quantite the signal.

2)

Next Generation Platform	Main advantages	Main disadvantages
Roche 454	Generate maximum read length	Expensive, high cost
Illumina	Suitable for whole genome sequencing, low cost, high throughput.	Lagging strand dephasing leads to the poor quality of read at the end.
Pacific Biosciences	Real time sequencing through single molecule approach generates maximum number of reads, low run time,	High cost, high error rate, low total number of reads
Oxford Nanopore	Low cost, long reads, minion is a usb device	Unknown error rates
Gilbert and sanger method of traditional sequencing	In Gilbert method, use of radioactive labels In Sanger method, fluorescent dye used for labelling.	Slow, high cost per run.