Mate-Paired library preparation / EcoP15I

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Scheme of Mate-Paired (EcoP15I) library preparation

Содержание


Presented scheme illustrates preparation of mate-paired fragments from long DNA. This part of the protocol is identical for Illumina and SOLiD platforms.


[править] DNA digestion

performed by ultrasound treatment (less than 1kb) or on Hydroshear (more than 1kb);


[править] Primary size selection

(optional) decrease a total amount of DNA, less enzymes will be used on the next steps;


[править] End-repair

three enzymes work simultaneously:

  • T4 DNA polymerase: digestion of 3' protruding ends;
  • Klenow DNA polymerase: extension of 3' recessive ends;
  • T4 PNK: phosphorilation of 5' ends & dephosphorilation of 3' ends, the best substrates are protruding and blunt ends.


[править] EcoP15I methylation

inactivates EcoP15I sites in genomic DNA (restriction enzyme will not recognize methylated sites). Reaction is not wery efficient, it is better to perform ON.


[править] Eco CAP adaptor ligation

ds adaptor contains EcoP15I recognition site; 100x molar excess to prevent ligation of DNA fragments with each other; it is better to keep ligation volume small, in large volumes circularization is preferable.


[править] Secondary size selection

proper size; removes adaptor dimers.


[править] Circularization in presence of Internal adapter

should be done at some optimal concentration: too low: self-circularization; too high: long concatemers. Double-stranded DNA is rigid, circularization impossible if fragment length <500bp.


[править] EcoP15 I restriction

    5'···CAGCAG(N)25-27···3'
    3'···GTCGTC(N)27-29···5'

structure of MP-fragments is presented here



[править] Next steps

are as in fragment library preparation:

Illumina

  • end-repair;
  • A-tailing;
  • adaptor ligation;
  • selection of biotin-harboring fragments;
  • preamplification.


SOLiD

  • end-repair;
  • adaptor ligation;
  • nick-translation;
  • selection of biotin-harboring fragments;
  • preamplification.


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