Use of real-time PCR for determining copy number and zygosity in transgenic plants

This review examines how real-time PCR can be used to determine copy number and zygosity in transgenic plants. Distinguishing between plants that harbor one and two copies of a transgene or are hemizygous and homozygous requires the ability to routinely distinguish twofold differences, a detection difference which approaches the resolution of PCR-based quantification methods. After explaining the basic principles, especially the threshold cycle (Ct value) as the basic measuring unit in real-time PCR, we introduce three quantitation methods currently in use. While the absolute and relative standard curve approaches are qualitative methods that distinguish high-copy from low-copy transformants, the comparative ( ) method with double-dye oligonucleotides (TaqMan probes) is able to detect twofold differences. In order to obtain reliable results, Ct values for an amplicon should be below 25 and the standard deviation below 0.3. Although real-time PCR can deliver exact copy number determinations, the procedure is not fail-safe. Therefore, real-time PCR should to be viewed as complementary to—rather than as a replacement of—other methods such as Southern analysis, but it is particularly useful as a preliminary screening tool for estimating copy numbers of a large number of transformants.     

Fingerprints and the determination of zygosity in twins

Zygosity was determined in 183 pairs of twins living in Aberdeen City, Scotland, by blood group studies, general appearance and fingerprints. The usefulness of simple methods of fingerprint analysis to determine zygosity was examined and it was found that comparisons using ridge counts were more efficient than those of pattern types.     

Tables for the diagnosis of zygosity in twins relative to hemogenetic polymorphisms

In order to determine the zygosity relative probabilities in favour of dizygotic twin pairs for 30 hemogenetic systems are given. The basic formulas for the probability calculations are described.     

Genetic analysis of dermatoglyphic patterns in twins.

Analysis of variance was performed on 71 dermatoglyphic variables in 424 twin sets. Using a method of twin analysis estimates of genetic variance were obtained. 54 of the variables were quantitated using a scoring system with modifications of arch or no pattern = 0, loops = 1, whorl = 2. The results indicated a significant genetic influence in most pattern areas. Patterning was more genetically controlled in the hand than in the foot. The hallucal area had the most significant genetic component of the foot while the patterns in the thumb had nonsignificant components of genetic variance. The thumb deviated from patterning in the rest of the fingers and may be more closely related to big-toe patterning.     

The influence of zygosity and chorion type on fat distribution in young adult twins consequences for twin studies.

An adverse intra-uterine environment has been associated with abdominal fat distribution in singletons. Twins often have a low birth weight and a short gestation. Therefore, they may have an increased risk to develop abdominal obesity. Furthermore, monozygotic monochorionic twins (MZ MC) have a larger intra-pair birth weight difference compared to monozygotic dichorionic twins (MZ DC). If adult anthropometry is programmed in utero, this may affect the intra-pair correlations in adulthood and, consequently, also the results from the classic twin method to estimate genetic and environmental influences. In the present study, we compared the absolute values, the intra-pair differences, and the intra-pair correlations of body mass, height, BMI, and abdominal fat distribution of 424 MZ MC, MZ DC and dizygotic (DZ) twin pairs (aged 18-34 yrs). DZ, MZ DC and MZ MC twins did not differ for most anthropometric characteristics. Only MZ women tended (p = 0.03) to accumulate more abdominal fat compared to DZ twins. Overall, the contribution of zygosity and chorion type to adult anthropometry was rather low (< or = 1.7%). Although the intra-pair birth weight difference of MZ MC pairs (10.5% in men, 12.3% in women) was significantly larger compared to that of MZ DC pairs (6.9% and 9.2% resp.), the intra-pair differences in adult anthropometry were similar for both MZ twin types. Also the intra-pair correlations of MZ MC and MZ DC pairs were strikingly alike, suggesting no significant influence of the prenatal environment on adult concordance. In conclusion, the substantial difference in the prenatal environment of MZ MC and MZ DC twins did not result in a difference in intra-pair concordance of adult anthropometry and fat distribution. Therefore, we suggest that the chorion type of MZ twins does not bias the twin design and the estimation of the genetic contribution to adult anthropometry.     

Use of immunosorbents for determining antibodies to histaglobulin]

A determination was made of antibodies to histaglobulin and gamma-globulin with the aid of immunosrobents in 178 sera of children suffering from allergic diseases, during the histaglobulin therapy. Results of investigations showed antibodies to histaglobulins to be absent in children untreated by this preparation. But they appeared in 57% of cases after the first course of histaglobulin treatment, and their incidence and their average level increased with an increase in the number of the courses of treatment carried out. gamma-Globulin antibodies were found at the initial condition in 42% of cases; this percentage rose to 68 after the first course of histoglobulin treatment. The authors believe that determination of histaglobulin antibodies during the treatment with this preparation could serve as an auxiliary immunological criterion of the efficacy of histaglobulin therapy.     

Use of a polarographic method for determining trichothecin]

The polarographic behaviour of trichothecin was studied. It was shown that the antibiotic could be detected in solutions at concentrations of 7.10(-7) moles with the help of the polarographic method. Conditions for the polarographic determination of trichothecin in fermentation broth were developed. The error was not more than 3 per cent. The reliability of the results was shown by statistical treatment of data performed in accordance with the requirement of the USSR State Pharmacopeia, X ed., prescribing that the precision of the assay is such that the fiducial limits at p = 95 per cent deviate from the average value by not more than 5 per cent. Comparison of the results of trichothecin determination in the fermentation broth with the polarographic and biological methods showed no significant difference. Therefore, the polarographic method may be recommended for trichothecin determination in the fermentation broth.     

Comparisons of dermatoglyphic patterns in monochorionic and dichorionic monozygotic twins

The data presented here indicate that different influences affect dermatoglyphic pattern development in MC-MZ and DC-MZ twins. Only five of 84 variables had significant mean differences but their clustering suggested a real difference in mean placement of the atd angle. Nineteen of 84 variables had significantly different within-pair mean squares for the two twin types. Larger numbers of twins will be required to obtain accurate estimates of the magnitude of the dermatoglyphic differences between MC-MZ and DC-MZ twins. Studies of dermatoglyphics in MC-MZ and DC-MZ twins are important to the understanding of factors which influence early embryonic development and when better documented may provide a mechanism for retrospectively diagnosing placental type of MZ twins.     

Race differences in the sex dimorphism of dermatoglyphic traits

For three dermatoglyphic traits it could be proved that there are racial differences in sex dimorphism. The results are as follow: (1) For the index of pattern intensity (158 samples), the sex difference is significantly higher in Negroids than in Europoids and Mongoloids. (2) For total ridge count, the sex difference is lower in Negroids than in Europoids. The discrepancy between (1) and (2) has been discussed. (3) The number of samples with higher frequencies of palmar patterns in males than in females is significantly higher in Negroids than in Mongoloids and Europoids for the hypothenar and III interdigital and lower in Mongoloids than in Europoids and Negroids for the II interdigital (max. 127 samples).     

A method for determining zygosity of transgenic zebrafish by TaqMan real-time PCR

When producing a genetically modified organism, intended genes are often integrated into a target genome by random insertions. Subsequently, it is often desirable to know the gene copy number of the transgenic organism and the zygosity of its offspring. Because of the random insertions, the estimation can be made only by quantitative measurement of the genes. Even though TaqMan real-time PCR has been used in gene expression analysis, it is routinely used to quantify differences larger than twofold or more than one PCR cycle. In this study, we employed TaqMan quantitative PCR to determine zygosity of transgenic fluorescent zebrafish in which a homozygote and a hemizygote differ by only twofold. We measured relative quantities of the transgene by taking the threshold cycle (Ct) of both the transgene and an internal control zebrafish genomic DNA. Using scatterplots and statistical inference, we demonstrated that homozygotes and hemizygotes could be differentiated unambiguously when multiple measurements were taken. We discuss the relationship between the repetitive measurements and TaqMan precision with a statistical model. The result illustrates that the method can be extended to some areas that require even higher precision such as determining the polyploidy of an organism.     

The diagnosis of zygosity in bull twins by means of the discriminant function for body conformation

Body conformation of monozygous twin bulls was considered as a variate, depending on single body measures. With the aid of a discriminant function, between-pair differences were maximized relative to within-pair differences. Two pairs of bull twins with identical blood types, which, nevertheless, gave rise to doubt of their monozygosity, could be discriminated unambiguously. Their within-pair variances for body conformation were at the between-pair level and half the between-pair level, respectively. The method was superior to a comparison of single body measures. The diagnosis of dizygosity reinforced arguments for dizygosity on the basis of behavioral and biochemical differences. The exact approach to body conformation is discussed in relation to multiple-factor analysis.175th Communication of the Research Institute of Animal Husbandry Schoonoord.     

Genetic analysis of bilateral dermatoglyphic asymmetry in twins and their parents

The data for this study were collected on 64 twin pairs (30 MZ and 34 DZ) and their 128 parents. Two following hypotheses were evaluated: 1. Bilateral asymmetry is significantly genetically controlled; 2. The twinning phenomenon would affect the magnitude of bilateral asymmetry. The results revealed no statistically significant differences between mean values of MZ and DZ twins and their parents for the majority of the traits. Significant differences were recorded for only 6 of 96 comparisons (6%). Analysis of variance revealed separated sources of MZ, DZ and singleton variance. F-ratios, contrasting variances between different groups were significant for 26 of 96 comparisons (27%) showing heterogeneity of variance between zygosities and between twins and their parents. In addition, environmental covariance appeared to be larger for MZ than DZ with respect to directional asymmetry (DA) for all 16 traits and fluctuating asymmetry (FA) for 14 traits. These observations showed complex environmental determinism for bilateral asymmetry for the majority of dermatoglyphic traits. Significant genetic variance ratios (GVRs) were observed for four variables (25%) with respect to DA and three variables (18.75%) with respect to FA. All these significant GVRs were rendered insignificant because of evidence of greater environmental covariance for MZ twins, except possibly for DA for URC4.     

The type of higher nervous activity in twins]

Exmination of twelve pairs of monozygote (MT) and dizygote (DT) twins and their control groups revealed an identity of the higher nervous activity type and a high positive intrapair and group correlation of typological parameters in MT (0.42); a predominantly different typological classification and a lower correlation in DT (0.33); a low correlation in the control group No. 1 and mere traces of general positive correlation in group No. 2 (0.07) and No. 3. The data point to the considerable genetic dependence of the higher nervous activity type; they also serve as a verification of the method used.     

Cross-cultural adaptation and preliminary validation of a zygosity determination questionnaire for twins in Sri Lanka.

We report the process of adaptation into Sinhala of a questionnaire given to mothers of twins to determine zygosity. Adaptation and validation was carried out in three stages. Firstly, we used a nominal group to translate the English version and to assess the extent of agreement (consensus measurement) on the appropriateness of the translation and resolve disagreement (consensus development). Secondly we used a qualitative interview with 25 mothers of twins. The three main stems of the translated questionnaire were used as a semi-structured interview, and the responses noted verbatim. These were categorised and analysed, and the translated full questionnaire was then presented as closed questions with fixed choice responses. The categorised responses generated during the qualitative interview were compared with the responses to the fixed choices in the full questionnaire. The third stage was the appraisal of the questionnaire by 17 bilingual parents of twins. The source and translated version of the questionnaire were given to them at least 3 days apart. The responses were rated and the total scores were computed to determine the zygosity. This step was carried out to measure the validity and reliability of the Sinhala version. A perfect correlation between the original and adapted version was obtained, with a kappa of 1. The results suggest that the Sinhala version of the questionnaire is conceptually equivalent to the original questionnaire. Comparison of the zygosity determination by using this adapted questionnaire with results from analysis of genetic markers on Sri Lankan twins is needed for final validation of the translated questionnaire.     

Why zygosity of multiple births is not always obvious: an examination of zygosity testing requests from twins or their parents.

This paper examines why parents of twins or adult twins themselves request zygosity testing. Of 405 multiples including 8 sets of triplets, the majority (93%) were monozygotic. Age of testing ranged from 0 days to 73 years. About 50% of requests came from parents or twins who were curious about, or expressed a need to be certain of, their zygosity. Other reasons included health concerns (current or future), other twins in the family, and misinformation about zygosity, frequently because of the erroneous assumption that all dichorionic twins are dizygotic. Parents of monozygotic twins may expect their twins to be 'identical' and believe their twins to be dizygotic because of minor phenotypic differences between them. Dizygotic twins like other siblings may share a phenotypic resemblance. Health professionals should be aware that zygosity of multiples may not always be obvious to parents and that accurate knowledge of zygosity may be justified.     

Use of paper indicator discs for determining the minimal inhibitory concentration of antibiotics]

Sensitivity of clinical strains of Staphylococcus and some Enterobacteriaceae to a number of widely used antibiotics was compared simultaneously with the use of two methods, i. e. the agar diffusion method and the method of serial dilutions. Regularities in distribution of the staphylococcal strains according to their sensitivity to antibiotics, such as erythromycin, benzylpenicillin, levomycetin and others were also studied with respect to every year using indicator paper discs. Interrelation observed during the comparison of the microbial sensitivity with the use of the two assay methods provided elaboration of the criteria for classification of the strains as "resistant" or "sensitive". The differentiation boarder for these two groups was determined according to the principle of the assay error minimization. A necessity of using standard dry media for specification of individual characteristics of various drugs in estimation of the microbial sensitivity to them by the agar diffusion method is emphasized.