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Home  |  Protocols  |  Molecular Biology  |  Inverse-PCR (I-PCR) for the identification of the breakpoints of deletions

Protocol - Inverse-PCR (I-PCR) for the identification of the breakpoints of deletions

The Inverse-PCR is a method that allows to amplify unknown regions of DNA, starting from the flanking known region on which are positioned the primers, in inverse direction compared to a normal PCR. It is necessary circularizing the DNA fragments, using ligase, then perform the I-PCR.

Inverse-PCR (I-PCR) for the identification of the breakpoints of deletions
Category:  Molecular Biology
Creation date: Mar 10, 2009
Last revision: Feb 08, 2016
Author(s):  Maria De Angioletti, Clementina Carestia, Giuseppina Lacerra
Contact
Name: Giuseppina Lacerra
Address: Institute of Genetics and Biophysics - CNR Via Pietro Castellino, 111 Naples 80131 ITALY
Phone: +39-081-6132602
Email: giuseppina.lacerra@igb.cnr.it

 

Figure legend:

Scheme of the Inverse-PCR method.

Steps

Description Temperature Time Note
Prepare tubes for I-PCR and add 10 ng of the circularized DNA (2 μl)
Prepare the I-PCR mix and dispense 23 μl in each tube with DNA sample
Prepare a 2% Nu Sieve GTG agarose gel (small) and load 12μl of PCR reaction. Also load a DNA ladder to check that the length of the amplicomer band
Recover the band and electro elute
Sequencing by dsDNA or by cloning

Other informations

I-PCR program

33 cycles:

1) T°: 94°              1 min

2) T°: 55°              1 min

3) T°: 72°              1.5 min

 

Quality validation: Yes

Validation info

The protocol has been used to define the breakpoints of two new deletions: the South-Italy β°-thalassemia and the Italian (G)γ((A)γδβ)°-thalassemia. The data has been published in peer-reviewed journals.

 

Citations
De Angioletti M, Sabato V, Musollino G, Prezioso R, Carestia C, Lacerra G. South-Italy β°-thalassemia: a novel deletion not removing the γ-globin silencing element and with 3' breakpoint in a hsRTVL-H element, associated with β°-thalassemia and high levels of HbF.Haematologica. 2013; 98(8): 98-100
Lacerra G, Prezioso R, Musollino G, Piluso G, Mastrullo L, De Angioletti M.Identification and molecular characterization of a novel 55-kb deletion recurrent in southern Italy: the Italian (G) γ((A) γδβ)°-thalassemia.Eur J Haematol. 2013;90(3):214-9

 

Related Protocols:
Related Oligos:

Solutions

  • Inverse-PCR mix

    Reagents F-concentration Quantity I-concentration
    Buffer
    1X
    2.5 μl
    10X
    Primer For
    0.4 μM
    10-12 pmol
    Primer Rev
    0.4 μM
    10-12 pmol
    dNTP
    0.2 mM
    4 μl
    1.25 mM
    Taq Polimerasi
    0.125 μl
    5 U/μl
    H2O bd
    up to 25 μl

    Note

    The primers used are in inverse direction

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