Protocol:DNA from amniocytes: phenol-chloroform method

DNA Preparation
The white blood cells (WBC) of peripheral blood are usually the most convenient source   of   human      genomic     DNA    for   DNA     analysis   with   respect   to haemoglobinopathies. It is estimated that 10 ml of whole blood yield approximately 250 &mu;g of DNA, more than sufficient for complete analysis of globin genes with the methods that are currently available (ie based on PCR). DNA is an extremely stable molecule, but enzymes which catalyse the breakdown of nucleic acids (nucleases) are found in all cells. In intact cells the DNA is found in the nucleus and thus is protected from the action of nucleases which are abundant in the lysosomes in the cytoplasm. However when cells are lysed, the membranes of the cell compartments are disrupted, allowing nucleases to come in to contact with the DNA. Thus the first stages of DNA extraction uses buffers which contain inhibitors of nuclease activity. Additionally all steps must be carried out at low temperatures (0oC). For long-term storage of samples prior to extraction a temperature of -70oC is recommended. There are many different methods described for DNA extraction from whole blood; the methods described below of salting–out and phenyl chloroform are used in the laboratories of some of the authors. There are also kits on the market for extracting DNA from blood samples which work well, but tend be expensive if used to prepare DNA from 5-10 ml blood samples.

DNA from amniocytes: phenol-chloroform method
The method of DNA extraction from amniotic fluid cells is essentially the same as for CVS, whether cells are from a direct sample or cultured. It is advisable to always have a culture set up as backup, because sometimes a direct fluid sample fails to provide sufficient amplifiable DNA to achieve a diagnosis. The cultured ells also provide confirmation of the result obtained on the direct cells, which are sometimes blood stained and the result open to possible doubt of from maternal contamination. The yield of DNA is smaller than that obtained from CVS, and therefore the amounts of all solutions used are one fifth of those for the CVS procedure (0.1 ml lysis buffer, 50 &mu;l of chloroform, etc.)

DNA from CVS: phenol-chloroform method
After sorting by microscopic dissection to remove all traces of maternal tissue, chorionic villi may be transported to the molecular diagnosis laboratory in either tissue culture medium or 0.5 ml of CVS lysis buffer. The latter is better if transportation is expected to take more than 24hours, as the DNA is perfectly stable at room temperature in CVS lysis buffer for up to two weeks or longer. This phenol extraction method is very quick, consisting of two 30 second phenol/chloroform extractions to remove protein, two chloroform washes to remove the phenol, and ethanol precipitation.

Reagents

 * 1) CVS Lysing solution: 150 mM NaCl, 25 mM EDTA, 0.1% SDS [Sodium dodecyl sulphate], 50 &mu;g/ml Proteinase K.
 * 2) Phenol (saturated with 0.1 M Tris) [Rathburn Chemicals]
 * 3) Chloroform
 * 4) 7.5 M Ammonium acetate
 * 5) 100% Ethanol (store in flammables cabinet with lock)
 * 6) 70% Ethanol

Method

 * 1) Transfer sorted CVS in tissue culture medium to a 1.5 ml Eppendorf tube. Spin at 3000 rpm for 30 sec and carefully remove the supernatant. If CVS is in lysis solution, go straight to step 4.
 * 2) Wash CVS by resuspension in 0.5 ml 150 mM NaCl, 25 mM EDTA. Spin at 3000 rpm for 30 sec and carefully remove the supernatant.
 * 3) Add 0.5 ml CVS lysing solution and mix by vortexing.
 * 4) Incubate over night at 37oC or at 55oC for 3 hours.
 * 5) Add 0.25 ml chloroform and 0.25 ml phenol (saturated with 0.1M Tris HCl). Mix and spin in a microfuge at 3000 rpm for 30 sec.
 * 6) Remove the upper aqueous layer to a clean appropriately labelled tube and carry out second extraction by repeating steps 5.
 * 7) Remove the upper aqueous layer to a clean appropriately labelled tube, add 0.5 ml chloroform and mix thoroughly.
 * 8) Spin at 3000 rpm for 30 sec and carefully transfer the supernatant to a clean labelled tube.
 * 9) Mix thoroughly and spin at 3000 rpm for 30 sec. Carefully transfer the supernatant to a clean labelled tube.
 * 10) Add 250 &mu;l of 7.5 M ammonium acetate and mix. Add 1.5 ml of 100% ethanol and gently mix to precipitate the DNA. This procedure should precipitate just DNA as a few white fibrous strands. If DNA is not observed at this stage, proceed to step 15
 * 11) Spin at 3000 rpm for 30 sec to pellet the DNA.
 * 12) Carefully pour off the 100% ethanol (taking care not to disturb the pelleted DNA) and add carefully 500 &mu;l of 70% ethanol to wash the DNA pellet. Spin at 3000 rpm for 30 sec.
 * 13) Carefully tip off the 70% ethanol wash leaving the DNA stuck to the bottom of the tube. Air-dry the pellet until all the ethanol has evaporated off it should still be clear.
 * 14) Dissolve the pellet in 50 &mu;l of sterile distilled water (use 25 &,u;l for small samples or 100 &mu;l for very large samples).
 * 15) If the DNA concentration is too low to see a precipitate at step 10, cool the ethanol mixture by placing it at -20oC for at least 1 hour, or by freezing it by submerging the tube in dry ice pellets for 5 min. Spin the tube at maximum speed in a microfuge for 20 min. Proceed from step 12. Note this procedure will precipitate both RNA and DNA and thus an accurate estimation of the DNA concentration cannot be made unless the RNA is removed by treatment with RNAase.