Human Sperm Preparation

1. Phosphate-buffered saline (PBS; Gibco BRL, Eragny, France).

2. 99% Methanol (Prolabo, Paris, France).

4. Glacial acetic acid (Prolabo).

5. 0.5 MNaOH (Merck Eurolab, Nogent-sur-Marne, France).

6. 20X Standard saline citrate (SSC): 3 MNaCl, 0.3 Mtrisodium citrate, pH 7.5 (this solution can be stored for several months at room temperature).

7. Washing buffer: 2X SSC diluted from 20X SSC.

Fig. 1. Principles of the multicolor PRINS reaction (modified from Yan [10J). Four sequential PRINS reactions, using specific centromeric primers for four chromosomes and three distinct fluorochrome-dUTPs (FITC-11-dUTP, TRITC-6-dUTP, Cascade Blue-7-dUTP) are performed without intermediate 3'-end blocking steps. The in situ mixing of the colors led to the final generation of four distinct colored spots (green, red, yellow, and blue), allowing the specific identification of each targeted chromosome. In this illustration, the chromosome 1 is successively labeled by the in situ incorporation of FITC, TRITC, FITC, and Cascade Blue, leading to a final yellow color. Chromosome 9 is labeled by FITC, TRIC, and Cascade Blue, leading to a red color. Chromosome 16 is labeled by the incorporation of FITC and Cascade Blue, resulting in a green color. Finally, chromosome 18 only incorporates Cascade Blue dUTP, giving a blue color. (Please see color insert following p. 48.)

Fig. 1. Principles of the multicolor PRINS reaction (modified from Yan [10J). Four sequential PRINS reactions, using specific centromeric primers for four chromosomes and three distinct fluorochrome-dUTPs (FITC-11-dUTP, TRITC-6-dUTP, Cascade Blue-7-dUTP) are performed without intermediate 3'-end blocking steps. The in situ mixing of the colors led to the final generation of four distinct colored spots (green, red, yellow, and blue), allowing the specific identification of each targeted chromosome. In this illustration, the chromosome 1 is successively labeled by the in situ incorporation of FITC, TRITC, FITC, and Cascade Blue, leading to a final yellow color. Chromosome 9 is labeled by FITC, TRIC, and Cascade Blue, leading to a red color. Chromosome 16 is labeled by the incorporation of FITC and Cascade Blue, resulting in a green color. Finally, chromosome 18 only incorporates Cascade Blue dUTP, giving a blue color. (Please see color insert following p. 48.)

8. Twin-frost glass microscope slides (CML, Nemours, France). The slides must be cleaned by soaking in absolute ethanol to which concentrated HCl has been added at the rate of 1 mL/100 mL. The slides are removed from the acid/alcohol and polished with a clean piece of muslin just before dropping the sperm suspension.

9. Light microscope Leica DMLB with x10, x40 magnification (Leica France, Rueil-Malmaison, France).

2.2. Multicolor PRINS

1. dATP: 100 mMsolution (Roche Diagnostics, Meylan, France) diluted 1:10 with sterile distilled H2O.

2. dCTP: 100 mM solution (Roche Diagnostics) diluted 1:10 with sterile distilled H2O.

3. dGTP: 100 mM solution (Roche Diagnostics) diluted 1:10 with sterile distilled H2O.

4. dTTP: 100 mM solution (Roche Diagnostics) diluted 1:100 with sterile distilled H2O.

5. 1 mMFluorescein isothiocyanate-12-dUTP (FITC-12-dUTP) (Roche Diagnostics).

6. 1 mM Tetramethylrhodamine isothiocyanate-6-dUTP (TRITC-6-dUTP) (Roche Diagnostics).

7. Cascade Blue-7-dUTP 1m M(Molecular Probes, Leiden, The Netherlands).

9. Bovine serum albumin (BSA; Sigma, St. Louis, MO).

10. Taq DNA polymerase (Roche Diagnostics) or AmpliTaq (Perkin Elmer, Foster City, CA).

11. 10X Taq buffer: 500 mM KCl, 100 mM Tris-HCl, pH 8.3, 15 mM MgCl2.

12. Oligonucleotide primers at 50 pmol/pL (see Table 1 and Note 1).

13. Sterile distilled water.

14. Tween-20 (Roche Diagnostics).

15. Washing buffer (diluted from 20X SSC): 4X SSC, 0.05% Tween-20.

16. 1.5-mL Sterile microcentrifuge tubes (Eppendorf AG, Hamburg, Germany).

19. Programmable thermal cycler equipped with a flat pate block (Hybaid Ltd., Teddington, UK).

2.3. Detection and Microscopy

1. 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI; Sigma).

2. Propidium iodide (Sigma).

3. Antifade solution Vectashield (Vector Labs, Burlingame, CA).

5. Rubber cement (Artos, Strasbourg, France).

6. Epifluorescence Microscope Leica DMRB (Leica France) equipped with x40 and x100 Plan FluoTar objectives, and with a DAPI single band-pass filter (Leitz filter A, cat. no. 513804), a FITC single band-pass filter (filter I3, cat. no. 513808), a TRITC single band-pass filter (filter N2.1, cat. no. 513812), a FITC/ TRITC double band-pass filter (filter G/R, cat. no. 513803) and a triple filter

Table 1

Characteristics of the Principal PRINS Primers Used in Human Cytogenetics

Table 1

Characteristics of the Principal PRINS Primers Used in Human Cytogenetics

Name

Locus

Chromosome

: Sequence

Annealing temp. (°C)

Optimal conc. (pmpl)

Alu S

alu

All

5'-GCCACTGCACTCCAGCCTGGG-3'

55

150

Alu R

alu

All

5'-GCCTCCCAACGTGCTGGGATTACAG-3'

55

150

Cons

asat

All

5'-TCTTTTTGTAGAATCTGCAAGTGGATA-3'

54

150

J52

sat III

1

5'-ATTCCATTAGATGATGACCCCTTTCAT-3'

61

100

1c

asat

1

5'-ATTCCATTAGATGATGACCCCTTTCAT-3'

61

100

2c

asat

2

5'-CTGTTCAACACTGRG-3'

71

150

3c

asat

3

5'-TGAGTTGAACACACACGTAC-3'

66

150

5c

asat

5

5'-TTCTGTCTAGCCTTACAGGAAAA-3'

70

150

7c

asat

7

5'-AGCGATTTGAGGACAATTGC-3'

56

100

8c

asat

8

5'-CTATCAATAGAAATGTTCAGCACAGTT-3'

67

150

9c

sat III

9

5'-AATCAACCCGAGTGCAATC-3'

56

150

10c

asat

10

5'-ACTGGAACGCACAGATGACAAAGC-3'

63

200

11c

asat

11

5'-GAGGGGTTTCAGAGCTGCT-3'

65

200

12c

asat

12

5'-GTTCAAATTCACAGAGTAT-3'

60

200

12A

asat

13

5'-TGATGTGTGTACCCAGCT-3'

60

100

16c

asat

16

5'-TTCTTTTCATACCGCATTCT-3'

53

75

17c

asat

17

5'-AATTTCAGCTGACTAAACA-3'

51

200

18c

asat

18

5'-ATGTGTGTCCTCAACTAAAG-3'

65

100

21A

asat

21

5'-TGATGTGTGTACCCAGCC-3'

61

150

Xc

asat

X

5'-GTTCAGCTCTGTGAGTGAAA-3'

68

75

D600

sat III

Y

5'-TCCATTCGATTCCATTTTTTTCGAGAA-3'

56

100

(filter B/G/R, cat. no. 513836) for simultaneous observation of DAPI/Cascade-Blue, FITC, and TRITC signals.

7. For image capturing, we use the software Metasystem Isis, Version 5.0 (Metasystem, Altlusshein, Germany).

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    What is a s a t chromosome?
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