The equipment, reagents and enzymes used in this study are listed in Appendix 1. The protocols for preparation of solutions can be found in Appendix 2.
Oligonucleotide primers were produced by Life Technologies. Primer sequences were obtained from Noble et al. (1994). The primers 5014 (5' CCC TTC CTG AGT GTC ATC A 3') and 971 (5' CGG CTG GCC AAG TTG TCT A 3') were used to amplify a 310 bp fragment spanning the polymorphic TaqI A site of the DRD2 gene.
Blood samples for use in this project were obtained from three sources:
Subjects were recruited by Dr Stanhope from among patients with an identified alcoholism problem. The subjects underwent a structured interview and formal diagnostic assessment. Subjects were all classified as alcohol dependent according to DSM-IV criteria, code 303.90 (Appendix 3).
Further samples were obtained from Dr Norton who had collected them for studies in pancreatitis. Samples were obtained from a group of known alcoholics without pancreatitis, and a group of known alcoholics with pancreatitis. All the alcoholics were classified as severe based on alcohol consumption data (>100 grams ethanol a day for >10 years).
A control population consisted of blood samples kindly obtained by Ms Pepper of the NSW Red Cross Blood Transfusion Service, Clarence Street Sydney.
Samples of blood that had previously been collected by Professor Po for ongoing polymorphic studies were used for comparison of the DRD2 allele frequency in a different ethnic population.
10 mL of sterile blood was collected from Dr Stanhope's Langton Clinic subjects. 3-5 mL of sterile blood was collected from Red Cross subjects. There was 1-3 mL of Burmese blood. All blood was collected in EDTA and stored at -20C for DNA analysis.
DNA was extracted from 1-10 mL of sterile unclotted blood using a salt extraction method, based on Miller, Dykes and Polesky (1988) (Appendix 4). Extracted DNA was dissolved in an appropriate amount of TE buffer. It was later quantified to check its concentration and quality (Appendix 5), and stored at -20C until required.
DNA amplification was carried out in 20 mL reactions using approximately 100 ng of genomic DNA (DNA concentration varied considerably, from as little as 10 ng to as much as 500 ng added per reaction), 50 ng of each primer and 0.5 U Taq DNA polymerase (5 units/mL) (Boehringer Mannheim and Pharmacia Biotech), 250 mM of each of the four dNTP's, 2.1-2.75 mM MgCl2, and 2 mL of the recommended buffer provided by the manufacturer (Boehringer Mannheim). Each reaction mixture was overlaid with 50 mL mineral oil to prevent evaporation during thermocycling.
Thermocycling was carried out on a Corbett Research FTS-320 Fast Thermal Sequencer. After an initial denaturation step at 96C for 3 minutes, the DNA was amplified in three step cycles:
A blank reaction was performed in each reaction, with a tube containing all the elements except DNA.
After each PCR was completed, the products were electrophoresed on a small agarose gel.
A simple method of preparing small agarose gels involves pouring 10 mL of 2% agarose (Appendix 2) onto 5 cm x 7.5 cm glass plates with the well combs held in place above the plates by clamps resting on the benchtop. Surface tension keeps the agarose from running off the sides. The plate and gel can then be placed into the buffer tank.
8 mL of PCR product was mixed with 1 mL of gel loading buffer and loaded into each well. A DNA molecular weight marker which contained pUC19 DNA digested with Hpa II was loaded in one lane. Electrophoresis was carried out at 60V for approximately 35 minutes - until the bromophenol blue marker dye had migrated 2/3 of the way down the gel.
The gels were stained in a 2 mg/L ethidium bromide staining bath for 10-15 minutes and photographed using a Gel Cam gel documenter (Bresatec, Australia).
Samples that had a good yield of PCR product as determined by electrophoresis were digested with the restriction enzyme Taq I (recognition sequence 5'-TCGA-3'). 10 mL of PCR product solution was digested with 4.5 U of Taq I enzyme (9 units/mL) in a 15 mL reaction mixture which also contained 1.5 mL of 10X reaction buffer provided by the manufacturer (Progen).
Digested DNA samples were mixed with 2 mL of gel loading buffer and loaded into 8 cm x 18 cm 5% polyacrylamide gels (Appendix 6) and electrophoresed (Hoefer Scientific Instruments) at 200V for 45 minutes - until the bromophenol blue marker dye had migrated to the end of the gel. Gels were stained in a 2 mg/L ethidium bromide staining bath for 15 minutes, visualised under UV light and photographed with Polaroid Film 667.
The acrylamide gels were then silver stained for four hours to overnight using the method of Neilan et al. (1994) (Appendix 7).
Individuals were genotyped as A1A1, A1A2 or A2A2 based on the pattern of banding as seen after polyacrylamide gel electrophoresis (Appendix 8).
In order to examine the extent of linkage disequilibrium within the DRD2 gene, analysis was begun on a second marker. The microsatellite marker used is located in the second intron of the DRD2 gene and has four common alleles of 80 (0.16), 82 (0.22), 84 (0.47), and 86 (0.15) base pairs (Hauge, 1991).
509 - 5'-CAG GAG CAC GTT TCT CAT AC-3' and
419 - 5'-GGA GGG CGG TGC GTT CAT-3' (Hauge, 1991)
PCR was performed in a total volume of 20 mL containing 1 mL of stock genomic DNA (50-500 mg/mL), 50 ng of each primer, 1 U Taq DNA polymerase (5 units/mL) (Boehringer Mannheim), 250 mM of each of the four dNTP's, 2.75 mM MgCl2, and 2 mL of the recommended buffer provided by the manufacturer (Boehringer Mannheim). Each reaction mixture was overlaid with 50 mL mineral oil to prevent evaporation during thermocycling.
After an initial denaturation step at 96C for 3 minutes, DNA was amplified in three step cycles of denaturation at 95C for 20 seconds, annealing at 58C for 30 seconds and extension at 72C for 60 seconds. After 35 cycles, the DNA was given a final extension step at 72C for 5 minutes.
PCR products were resolved on 2% agarose gels to check for quality of PCR product. The samples that amplified well were then electrophoresed on 18 cm x 16 cm 8% polyacrylamide gels at 200 V for 2 hours. Gels were stained in a 2 mg/L ethidium bromide staining bath for 15 minutes, visualised under UV light and photographed with Polaroid Film 667. The gels were then silver stained (Appendix 7).
Each population was tested for conformity to Hardy-Weinberg equilibrium. Hardy-Weinberg equilibrium is used to demonstrate the hypothesis that two codominant autosomal alleles, in this case A1 and A2, is the most likely genetic theory to explain the three phenotypes (A1A1, A1A2 and A2A2) seen in the population. The Hardy-Weinberg principle states that the genotypic frequencies for a gene with two different alleles are a binomial function of the allelic frequencies (Weaver and Hedrick, 1991). It assumes that mutation, genetic drift, gene flow, and selection are not affecting genetic variation. If the A1 allele frequency is represented by p, and the A2 allele frequency is represented by q, then the expected number for each phenotype is as follows:
A1A1 - p2n
A1A2 - 2pqn
A2A2 - q2n
where n is the total number of alleles.
Conformity to Hardy-Weinberg equilibrium is tested using a chi-2 test between observed and expected numbers to determine whether the observed numbers are consistent with Hardy-Weinberg predictions. In this case, the number of degrees of freedom is the number of phenotypes minus the number of alleles, or 3 - 2 = 1.
The number of columns in a contingency table is denoted by c and the number of rows by r, and there are r x c cells in the table. The null hypothesis for contingency table testing is that the frequencies of observations found in the rows are independent of the frequencies of observations found in the columns (or, that the column frequencies are independent of the row frequencies) (Zar, 1984).
chi-2 values are calculated using the formula
chi-2 = S (O-E)2
E
Where O is the observed number and E is the expected. Expected values for each cell are calculated using the formula:
E = R x C
T
where R is the total of the row the cell is in, C is the total of the column the cell is in, and T is the sum of all cells. The degree of freedom is the number of rows minus one times the number of columns minus one:
df = (r-1)(c-1)
The chi-2 test using contingency tables was used to compare differences in phenotype distribution, allele prevalences and allele frequencies between the Red Cross, Langton Clinic and Burmese populations, as well as between the current populations and others' populations.
If we wish to combine data from more than one source, it is possible to perform a heterogeneity test using chi-square analysis (Zar, 1984). This allows the investigator to establish that the samples came from the same effective population, and so whether it is valid to combine the results for further analysis. Heterogeneity among alcoholics and controls can be studied with a chi-2 test using 3 x n tables and (n-1)(3-1) or degrees of freedom, where there are three phenotypes, and n is the number of studies. If the assumption that the samples came from the same population is true, then the samples are homogeneous. If it is not true, then the samples are said to be heterogenous and the chi-square analysis on the pooled data would not be justified (Zar, 1984). The heterogeneity chi-square was performed on studies to be used for meta-analysis.