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Home  |  Protocols  |  Molecular Biology  |  Circularization of DNA fragments for the molecular characterization of new deletions

Protocol - Circularization of DNA fragments for the molecular characterization of new deletions

During the molecular characterization of new deletions (to define the breakpoints) can occur that one extreme is known, but not the second one. In these cases it is very useful and suitable to perform the circulatization by ligase of DNA fragments containing the beakpoints of the deletion, followed by the inverse-PCR. Using these methods we were able to define the breakpoints of two new beta-Thalassemia deletions.

Circularization of DNA fragments for the molecular characterization of new deletions
Category:  Molecular Biology
Creation date: Jan 13, 1997
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:

(A) The structure of the β-globin gene cluster resulting from the ~66 kb deletion (South-Italy β-thal). An arrow indicates the position of breakpoint. (B) Restriction mapping of the breakpoint region of the deletion. Vertical arrows indicate the position of the breakpoint. The expected length of the normal restriction fragments is reported on the left. The 4.2 kb abnormal Pvu II restriction fragment used for the Inverse PCR is highlighted by a box. The horizontal arrows show the position of the primers used for the I-PCR (De Angioletti et al. 2013).

Steps

Description Temperature Time Note
Select the restriction enzyme giving rise to an anomalous pattern through restriction mapping analysis
1) Digestion of DNA from a carrier of the new deletion with the selected restriction enzyme following the protocol reported below
over night
Prepare 1.5 ml tube for each reaction and the reaction mix as reported below
2) Prepare a 1.2% GTG Nu-Sieve agarose gel in 1X TBE
Choose a beaker that is 2-4 times the volume of the solution
Add the cold 1X TBE solution and a stir bar, than slowly spill the agarose powder while the solution is stirred. Cover the beaker with aluminum sheet
Place on heated plate and boil while stirring until the agarose is dissolved
10 minutes
Add an intercalant of the DNA (ethidium bromide or other). Allow to cool on cold plate while stirring
10 minutes
Pour the agarose into the tray with comb. Allow to cool, and cover with foil or plastic wrap and hold
cold room
over night
3) Electroforesis of digested DNA on agarore gel (1.2% NuSieve GTG agarose gel)
Load the samples of DNA between two DNA ladders, chosen in according to the length of the fragment to be recovered. Migrate to 22mA
9 hours
4) Excision of DNA band from gel
Cut out the region of the gel in correspondence to the band of interest, using as reference the DNA ladders bands
Put the recovered band in a 1.5 ml tube
Melt the gel
65° C
10 minutes
Define the volume of the liquid agarose. Add 1X TE so that the final concentration of agarose is <0.5%
65° C
10 minutes
5) Extraction and precipitation of DNA from agarose gel
Add one volume of phenol saturated with TE
Shake and centrifuge at 10.000g
RT
10 minutes
Transfer the upper aqueous phase to a clean tube
Add one volume of chloroform-isoamyl alcohol 24:1, shake and centrifuge at 10.000g
RT
10 minutes
Recover the upper phase and determine the volume
Add sodium acetate 3.3M, coming to a final concentration of 0.33M, and two volumes of 95% cold ethanol
Centrifuge at 12.000g
4° C
30 minutes
Remove the supernatant paying attention to the pellet
Wash the DNA pellet with 500μl of 80% cold ethanol
-20° C
Centrifuge at 12.000g
4° C
30 minutes
Discard the supernatant and dry the DNA pellet in a vacuum concentrator (Savant) or at RT
Suspend the DNA pellet in 50μl TE
Prepare a 1% agarose (normal) gel
Load 5μl of the recovered DNA and DNA ladders for a quantitative and qualitative analysis
Determine the amount of the recovered DNA
6) Circularization of DNA fragment by DNA ligase
For each ligase prepare a 1.5ml tube as follows:
-DNA digested with R.E. in TE 100ng
-H2O bd up to 150μl
To facilitate the distension of the digested DNA fragments incubate
65° C
10 minutes
after incubate put in ice with H2O
Add into each tube the 50 μl of the Ligase mix
Centrifuge and incubate
16° C
over night
To inactivate theT4 DNA Ligase incubate
65° C
10 minutes
7) Purification of DNA circularized
Add one volume of phenol-chloroform, shake and centrifuge at 12.000g
RT
10 minutes
Transfer the upper aqueous phase to a fresh tube and precipitate with the addition of 20μl of 3.3M sodium acetate and 2 volumes (400μl) of 95% ethanol
Incubate
-80° C
30 minutes
Centrifuge at 12.000g
4° C
10 minutes
Wash with 80% ethanol
Centrifuge as above and drying in a vacuum concentrator(Savant)
Re-suspend in 20μl TE 1X
Proceed with the Inverse-PCR protocol

 

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

 

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Solutions

  • Restriction Enzyme reaction mix (40μl)

    Reagents F-concentration Quantity I-concentration
    DNA
    15 μg
    Restriction enzyme
    1 μl
    20U/μl
    Buffer 10X
    4 μl
    10X
    H2O
    up to 40 μl

  • Ligase mix (50 μl)

    Reagents F-concentration Quantity I-concentration
    Buffer
    20 μl
    10X
    T4 DNA Ligase
    1 μl
    400.000 U/ml
    H2O bd
    29 μl

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