mRNA Library Prep: cDNA clean up through adapter ligation - - PowerPoint PPT Presentation

mRNA Library Prep: cDNA clean up through adapter ligation

Reagents

Their Role in the Reaction

 Deoxynucleotide Mix  T4 DNA Polymerase  E. coli DNA Polymerase I,

Large (Klenow) Fragment

 T4 Polynucleotide Kinase



T4 DNA polymerase and Klenow enzyme converts overhangs into blunt ends



T4 is a bacteriophage of E. coli.



3ʹ to 5ʹ exonuclease activity of enzymes removes 3ʹ overhangs and polymerase activity fills in 5ʹ

• verhangs.



T4 PNK phosphorylates 5ʹ ends of DNA fragments

Reagents

Their Role in the Reaction

 Deoxyadenosine 5 ́-

Triphosphate (1 mM dATP)

 Klenow Fragment (3 ́→5 ́

exo–)

 Prevents fragments from

ligating to one another during adapter ligation reaction

 Corresponding single ‘T’

nucleotide on 3’ end of adapter provides a complementary overhang for ligating adapter to fragment

 To ensure a low rate of

chimera (concatenated template) formation

Reagents

Their Role in the Reaction

 DNA Adaptors (15 µM)  Quick T4 DNA Ligase

 Adds distinct sequences to the

5’ and 3’ ends of each strand in the genomic fragment

 Additional sequences added by

tailed primers during PCR

 Additional sequences are

necessary for library amplification on flow cell during cluster formation