DNA Diagnostic Laboratory - Human and Clinical Genetics (LUMC) - LMDp_Protocols©

DNA isolation from chorionic villi

(last modified December 30, 1999)

Protocol

for the isolation of DNA from leucocytes (kindly provided by Bert Bakker and Els Voorhoeve)

Target group

The technicians who are working in the clinical DNA-diagnostic department.

Responsibilities

The technicians responsible for the diagnostic analysis of the chorionic villi are also responsible for the isolation of DNA from the villi.

Definitions

Description

This protocol describes the method used to isolate DNA from chorionic villi. Lysis of the villi is always followed by a phenol/chloroform extraction. Chorionic villi are aspirated in the 10-13th week of the pregnancy. The chorionic villi sample is examined at the department of obstetrics and split in two portions. One part of the chorionic villi sample is karyotyped by a cytogenetic department, the other part is sent to the laboratory for DNA-diagnosis. When the sex of the foetus is determined, we receive a phone call from the genetic counsellor or the cytogeneticist.

This protocol is used when for prenatal diagnosis the Southern blot method (ALG004) has to be performed. If this is not the case, the DNA isolation from chorionic villi is performed according to ALG026 using the QIAamp® DNA Mini Kit.

The receipt of all the villi samples and their processing takes place in the pre-PCR lab. Storage of the DNA-samples is in the cold room. DNA-samples should never enter the post-PCR rooms.

Durables

NOTE: equivalent durables may be used

Consumables

NOTE: equivalent consumables may be used

Chemicals

NOTE: equivalent chemicals may be used

Solutions

For the solutions marked with a * a special form (with the protocol on it) is filled out. The other solutions are ready to use or made as described here.

Rules for the DNA-isolation shift

General

Administration

DNA isolation protocol

  1. Day 1
    Starting material for this isolation protocol are chorionic villi in culture medium
    NOTE: do not throw away the culture media in which the villi were sent and the wash solutions before you have isolated (enough) DNA on day two
    1. fill out the WCHOR-form
    2. transfer all the villi and the medium to a 50 ml blue cap tube, rinse the bottle and the cap with 1 x SE-buffer to ensure that all the villi are transferred
    3. fish out all the villi and transfer them to a clean 50 ml blue cap tube with a (large) Pasteur pipet (keep the villi in the capillary part of the pipet otherwise they will stick to the glass), add 1 x SE-buffer to a final volume of ± 40 ml and wash the villi by inverting the tube several times
    4. give a description of the villi and ask for a second opinion
    5. remove blood clots and other debris and isolate DNA out of these "dirty" villi separately; generate a new D1 isolation number for this sample in Oracle
    6. transfer the clean villi to a cryotube in a final volume of 0.5 ml of 1 x SE-buffer
      NOTE: if you have received a large quantity of villi it is better to split them in two aliquots (generate a new D1 isolation number for a second sample in Oracle)
    7. add 75 µl pronase (20 mg/ml) and mix gently
    8. add 70 µl 10% SDS
    9. degrade the villi by placing them on the rollerbench for 30 min, followed by an overnight incubation in the 37°C waterbath
  2. Day 2
    CAUTION: phenol can cause severe burns to skin and damage clothing. Gloves and a lab coat are to be worn whenever working with phenol, and all manipulations are carried out in a fume hood. Discard all phenol containing solutions in the appropriate organic waste (tank with red label)
    1. check if the solution has become clear and viscous. If this is not the case add extra pronase and put the tubes on the rollerbench for 2-3 hours
    2. when the villi are well degraded, a phenol:chloroform:iso-amyl alcohol extraction [2] is performed; using a P200 with a Cell Saver tip, transfer the clear and viscous DNA-containing solution to an 1.5 ml eppendorf tube with
    3. estimate the volume of the solution, add an equal volume of phenol:chloroform:iso-amyl alcohol and put the tube(s) in the topover for 10 min
    4. centrifuge for 10 min at 14,000 rpm
    5. using a P200 pipet with a Cell Saver tip, transfer the upper aqueous phase into an 1.5 ml eppendorf tube
      NOTE: avoid the transfer of material from the interface
    6. repeat steps 3, 4 and 5 if a lot of debris is visible in the interface. Make a remark of this repetition in form WCHOR
    7. add an equal volume of dichloroform:iso-amyl alcohol, mix the phases by inverting the tube(s) for 30 sec
    8. centrifuge for 5 minu at 14,000 rpm and transfer the upper aqueous phase to an 1.5 ml eppendorf tube without disturbing the interface
    9. add 1/20 volume of 3.0 M NaAc (pH5.6) and mix by inverting the tube
    10. precipitate the DNA with one volume of 2-propanol
    11. fish out the precipitated DNA with a sealed Pasteur pipet
    12. wash the DNA in 70% ethanol
    13. dissolve the DNA in 100-200 µl TE-4 in a Nunc Cryotube with a printed label (see administration)
    14. to inactivate possible DNases, incubate the tube for 10 min in a 65°C waterbath
    15. put the tube(s) on the rollerbench for several hours or overnight (RT)
    16. store the dissolved DNA sample(s) in the DNA storage refrigerator
    17. complete the WCHOR-form and store it in the folder WCHOR

References

[1] Bakker E, Kneppers ALJ, Voorhoeve E, Deutz-Terlouw PP, Bröcker-Vriends AHJT, van Ommen GJB (1991). Advances and pitfalls in prenatal diagnosis: Five years DNA-analysis for Duchenne and Becker muscular dystrophy and haemophilia. Muscular Dystrophy Research C. Angeline et al editors. Elsevier 1991, p67-76.

[2]Ausubel F.M., Brent R., Kingston R.E., Moore D.D., Seidman J.G., Smith J.A., Struhl K. (1987) Current Protocols In Molecular Biology. Volume 1. Wiley Interscience, Chapter 1.2.1

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