Klausur GR

ingredients

• DNA polymerase, primer, template DNA

• chain terminating nucleotide each labled with a unique color of dye -> ddNTPs (Dideoxynucleotides)

Steps

• combine ingredients

• denaturation

• cooling and primer binding to single strand

• DNA polymerase binding and synthezising complete strand

• termination by ddNTP

• repeat process

• gel electrophoresis -> laser can detect unique color -> Chromatogram

uses and limitation

• high quality

• for individual pieces of DNA

• Expensive and inefficient

workflow

1. library preparation

   • prepared by random fragmantation of the DNA or cDNA sample

   • 5’ and 3’ adapter ligation

   • fragments are PCR amplified and gel purified

2. cliuster generation

   • library loaded onto a flow cell

   • fragments are captured on a lawn of surface bound oligos

   • bridge amplification -> distinct clonal clusters

3. sequencing (reversible termination method)

   • sequencing reagents are added

   • first base gets incorporated -> flow cell gets imaged -> emission recorded -> cycle repeated

4. alignment and data analysis

   • reads are aligned to a reference sequence

problem

• only short reads

• must be assembled to reference

• difficult for repetitive sequences