Biology METHODOLOGIES

KEY TOPICS

star Two major approaches
star Methods in detail

TWO MAJOR APPROACHES

● The methods involved in HGP used color{Violet}"two major approaches".

● One approach focused on identifying all the color{Violet}"genes" that expressed as color{Violet}"RNA" (referred to as color{Violet}"Expressed Sequence Tags" (color{Violet}"ESTs").

● The other took the color{Violet}"blind approach" of simply sequencing the color{Violet}"whole set of genome" that contained all the color{Violet}"coding and non-coding" sequence.

● And later it assigned color{Violet}"different regions in the sequence" with functions (a term referred to as color{Violet}"Sequence Annotation").

METHODS IN DETAIL

● For sequencing, the color{Violet}"total DNA" from a cell is isolated and converted into color{Violet}"random fragments" of color{Violet}"relatively smaller sizes" (since DNA is a very long polymer, and there are technical limitations in sequencing very long pieces of DNA).

● It is then color{Violet}"cloned" in suitable host using color{Brown}"specialised vectors".

● The cloning resulted into color{Violet}"amplification of each piece" of DNA fragment so that it subsequently could be sequenced with ease.

● The commonly used hosts were color{Violet}"bacteria and yeast", and the vectors were called as color{Violet}"BAC" (color{Violet}"bacterial artificial chromosomes"), and color{Violet}"YAC" (color{Violet}"yeast artificial chromosomes").

● The fragments were sequenced using color{Violet}"automated DNA sequencers" that worked on the principle of a method developed by color{Violet}"Frederick Sanger".

● These sequences were then arranged based on some color{Violet}"overlapping regions" present in them.

● This required generation of color{Violet}"overlapping fragments" for sequencing.

● color{Violet}"Alignment" of these sequences was color{Violet}"humanly not possible".

● Therefore, color{Violet}"specialized computer" based color{Violet}"programs" were developed.

● These sequences were color{Violet}"subsequently annotated" and were assigned to each color{Violet}"chromosome".

● The sequence of color{Violet}"chromosome 1" was completed only in color{Violet}"May 2006" (this was the color{Violet}"last of the 24" human chromosomes – 22 autosomes and X and Y – to be sequenced).

● Another challenging task was assigning the color{Violet}"genetic and physical maps" on the genome.

● This was generated using information on color{Violet}"polymorphism" of color{Violet}"restriction endonuclease" color{Violet}"recognition sites", and some repetitive DNA sequences known as color{Violet}"microsatellites".