Which step is done first in map-based sequencing?
Map-based or clone-by-clone sequencing strategy, used for human genome project, first establishes a low-resolution physical map of genome. Initial step involves sizing large genomic fragments created by complete digestion with rare-cutting enzymes using pulsed-field gel electrophoresis, which resolves megabase-sized DNA through alternating electric fields. This allows ordering of yeast artificial chromosome and bacterial artificial chromosome clones into contigs based on overlapping fingerprints before subcloning into smaller vectors and shotgun sequencing. Early mapping prevents misassembly and provides chromosomal anchoring absent in whole-genome shotgun approach.
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.