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NCERT Exemplar · Q69

Q.Give an account of the methods used in sequencing the human genome.

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The Human Genome Project sequenced the roughly 3-billion-base-pair human genome using two complementary strategies — Expressed Sequence Tags (identifying only genes that are expressed as RNA) and sequence annotation (sequencing the entire genome, coding and non-coding, then assigning function to different regions afterward) — both built on cloning DNA fragments into BAC/YAC vectors, automated Sanger sequencing, and computer-assisted assembly of overlapping fragments.

Sequencing an entire genome is never done by reading straight through 3 billion base pairs in one continuous pass — no sequencing technology works that way. Instead, the genome first has to be broken into much smaller, manageable pieces, each piece sequenced individually, and the resulting short sequences reassembled by finding where they overlap. The Human Genome Project (HGP) used two broad methodologies to decide what to sequence and in what order.

The first methodology is based on Expressed Sequence Tags (ESTs). Here, researchers identify all the genes that are actually expressed as RNA in a cell by sequencing complementary DNA (cDNA) made from that mRNA. Because this approach only ever looks at sequence that corresponds to a real, functioning gene, it is a faster and more targeted way of locating genes — it simply ignores the large stretches of non-coding DNA that lie between genes.

Note

The NCERT textbook describes the EST approach and the whole-genome "sequence annotation" approach as the two broad methodologies actually used to sequence the human genome, alongside the physical/genetic mapping techniques used to order the resulting fragments along each chromosome.

The second methodology, sequence annotation, takes the opposite approach: instead of only sequencing expressed genes, it sequences the whole set of the genome — both the coding sequences and the much larger non-coding regions in between. Only after this complete sequence is obtained are different stretches of it annotated, or assigned a function, based on further analysis. This route is far more exhaustive and required considerably more sequencing effort than the EST approach, but it is what ultimately produced a complete reference sequence of the human genome rather than just a catalogue of genes. …

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