What is the need of "cluster preparation " ?

[sivasish]

Hi,

What is the use of "cluster generation /library preparation" step in Next generation sequencing ? Can't we squence the fragments after ligating fragments to primers on flowcell ? (Illumina )

[cliffbeall]

The cluster generation step is to amplify the single molecules to many in a small area so they can generate a detectable fluorescent signal.

[sivasish]

Hi ,Thanks much !!. so ... a cluster instead of individual/single fragment is bombarded with four fluorescent labelled nucleotides at a time and the emitted color with higher percentage is detected and considered.

plz ,correct me if I got it wrong.

[protist]

The nucleotides are not not radiolabelled, they carry a fluorescent label .

[sivasish]

oops..sry..thanks !! Can u tell me whether my assumption is right or wrong ?

[pmiguel]

There is no "bombardment". An oligo is annealed to a fixed position in the adapters of all the clusters. This oligo is then extended, one base at a time, with fluorescently labelled dNTPs. After each base incorporation all the nascent strands in a cluster will glow a certain color specified with the base that was incorporated. (A different dye is attached to each kind of dNTP). T\he flowcell is scanned to read the color of each cluster. Then the dye is removed from all the terminal bases in a process that also "unblocks" that base so it can be extended in the next cycle.

--
Phillip

[sivasish]

Thanks much !! .. I got it..