Isolation of genomic DNA from milk samples by using Chelex resin

1997 ◽  
Vol 64 (2) ◽  
pp. 231-238 ◽  
Author(s):  
MARCEL AMILLS ◽  
OLGA FRANCINO ◽  
MARTA JANSA ◽  
ARMAND SANCHEZ
Keyword(s):  
Genomic Dna ◽  
Bovine Genome ◽  
Critical Factor ◽  
Milk Composition ◽  
Exon 2 ◽  
Casein Gene ◽  
Milk Samples ◽  
Chelex Resin ◽  
Drb Gene ◽  
Number Of Cycles

A rapid procedure for isolating genomic DNA from milk samples has been devised, based on the use of Chelex resin. By using this protocol, genomic DNA was extracted from milk samples from 15 cows and 15 goats. The suitability of these DNA preparations as a template for performing the polymerase chain reaction (PCR) was tested by amplifying three different loci of the bovine genome: exon 4 of the κ-casein gene and the INRA5 and INRA23 microsatellites, together with two others: exon 19 of the αs1-casein gene and exon 2 and part of intron 2 of the DRB gene of the caprine genome. No amplification products could be obtained from any samples at 30 cycles. In contrast, at 45 cycles the number of amplified samples ranged from 86 to 100% and at 65 cycles all the DNA targets were amplified, indicating that the number of cycles was a critical factor to be optimized for obtaining the desired PCR target. These results suggest that this method may be a useful tool for analysing genetic polymorphism at the DNA level by PCR and relating it to milk composition and other traits of economic interest.

scholarly journals Human Milk Composition and Dietary Intakes of Breastfeeding Women of Different Ethnicity from the Manawatu-Wanganui Region of New Zealand

Nutrients ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 1231 ◽  
Author(s):  
Christine Butts ◽  
Duncan Hedderley ◽  
Thanuja Herath ◽  
Gunaranjan Paturi ◽  
Sarah Glyn-Jones ◽  
...  
Keyword(s):  
Fatty Acids ◽  
New Zealand ◽  
Breast Milk ◽  
Human Milk ◽  
Milk Composition ◽  
Pacific Island ◽  
Dietary Intakes ◽  
Omega 3 ◽  
Food Diary ◽  
Milk Samples

Human milk is nutrient rich, complex in its composition, and is key to a baby’s health through its role in nutrition, gastrointestinal tract and immune development. Seventy-eight mothers (19–42 years of age) of Asian, Māori, Pacific Island, or of European ethnicity living in Manawatu-Wanganui, New Zealand (NZ) completed the study. The women provided three breast milk samples over a one-week period (6–8 weeks postpartum), completed a three-day food diary and provided information regarding their pregnancy and lactation experiences. The breast milk samples were analyzed for protein, fat, fatty acid profile, ash, selected minerals (calcium, magnesium, selenium, zinc), and carbohydrates. Breast milk nutrient profiles showed no significant differences between the mothers of different ethnicities in their macronutrient (protein, fat, carbohydrate, and moisture) content. The breast milk of Asian mothers contained significantly higher levels of polyunsaturated fatty acids (PUFAs), omega-3 (n-3) and omega-6 (n-6) fatty acids, docosahexaenoic acid (DHA), and linoleic acids. Arachidonic acid was significantly lower in the breast milk of Māori and Pacific Island women. Dietary intakes of protein, total energy, saturated and polyunsaturated fat, calcium, phosphorus, zinc, iodine, vitamin A equivalents, and folate differed between the ethnic groups, as well as the number of serves of dairy foods, chicken, and legumes. No strong correlations between dietary nutrients and breast milk components were found.

scholarly journals Novel LNA probe-based assay for the A1 and A2 identification of β -casein gene in milk samples

2021 ◽  
pp. 100055
Author(s):  
Rodrigo Giglioti ◽  
Cintia Hiromi Okino ◽  
Bianca Tainá Azevedo ◽  
Gunta Gutmanis ◽  
Luciana Morita Katiki ◽  
...  
Keyword(s):  
Casein Gene ◽  
Milk Samples ◽  
Lna Probe

Effect of mid-line or low-line milking systems on lipolysis and milk composition in dairy goats

2018 ◽  
Vol 156 (6) ◽  
pp. 848-854
Author(s):  
M. C. Beltrán ◽  
A. Manzur ◽  
M. Rodríguez ◽  
J. R. Díaz ◽  
C. Peris
Keyword(s):  
Fatty Acid ◽  
Free Fatty Acid ◽  
Milk Composition ◽  
Crossover Design ◽  
Dairy Goat ◽  
Dairy Goats ◽  
Milk Samples ◽  
Commercial Farms ◽  
Milk Storage ◽  
Enzymatic Coagulation

AbstractTwo experiments were carried out to investigate how milking in mid-line (ML) affects the lipolysis level and milk composition in goat livestock, in comparison with low-line (LL) milking. The first experiment took place, in triplicate, on an experimental farm. For each replicate, a crossover design (62 goats, two treatments, ML and LL, in two periods each lasting 4 days) was used. Milk samples were taken daily at 0 and 24 h after milking. In the first experimental replicate, some enzymatic coagulation cheeses were made, which were assessed by a panel of tasters at 50 and 100 days of maturation. In the second experiment, the lipolysis level and composition of tank milk from 55 commercial dairy goat farms (25 ML and 30 LL) were analysed, in milk samples taken in three different weeks. The results of the first experiment showed that ML milking increased free fatty acid (FFA) concentration in raw goat's milk significantly (0.71 v. 0.40 mmol/l, respectively). However, in the milk samples taken from commercial farms the FFA concentration remained unaffected by the milking pipeline height (0.59 v. 0.58 mmol/l for ML and LL, respectively). No significant differences were found in the milk composition, nor in the sensory characteristics in the cured cheeses, which suggests that factors other than the milkline height are able to influence the level of lipolysis under commercial conditions. Therefore, ML milking should not be discouraged, provided that the correct functioning and management of the milking operation and milk storage on the farm is guaranteed.

scholarly journals Determination of Fat, Protein, and Lactose in Raw Milk by Fourier Transform Infrared Spectroscopy and by Analysis with a Conventional Filter-Based Milk Analyzer

1996 ◽  
Vol 79 (3) ◽  
pp. 711-717 ◽  
Author(s):  
Dominique Lefier ◽  
Remy Grappin ◽  
Sylvie Pochet
Keyword(s):  
Fourier Transform ◽  
Fourier Transform Infrared ◽  
Raw Milk ◽  
Milk Composition ◽  
Milk Samples ◽  
Bulk Milk ◽  
Different Seasons ◽  
Standard Deviations ◽  
True Protein ◽  
Single Calibration

Abstract The accuracy of fat, crude protein (CP), true protein (TP), and lactose determinations of raw milk by Fourier transform infrared (FTIR) spectroscopy and by analysis with a conventional filter-based milk analyzer was assessed in 6 trials performed over a 10-month period. At each trial, 30 bulk milk samples collected from 15 European countries and 11 reconstituted milks made from raw milk components were analyzed. When calibrations were performed with reconstituted milks at each trial, accuracy standard deviations for fat, CP, TP, and lactose were, respectively, 0.050,0.048,0.035, and 0.076 g/100 g for the filter instrument and 0.047, 0.046,0.042, and 0.065 g/100 g for the FTIR instrument. When a single calibration was made instead of calibrations at each trial, accuracy standard deviations increased for the filter instrument to 0.130, 0.119,0.121, and 0.083 for fat, CP, TP, and lactose, respectively, and for the FTIR instrument to 0.082, 0.053,0.044, and 0.084 g/100 g. Because the FTIR instrument provides more spectral information related to milk composition than does the filter instrument, single-calibration FTIR analysis of milk samples collected in different seasons is more accurate. Using reconstituted milks, prepared such that there is no correlation between fat, CP, and lactose, provides a more robust calibration than using genuine bulk milk, especially when milks with unusual composition are analyzed.

scholarly journals Effect of ewes entry order into milking parlour on milkability and milk composition

2017 ◽  
Vol 62 (No. 9) ◽  
pp. 392-402 ◽  
Author(s):  
L. Mačuhová ◽  
V. Tančin ◽  
J. Mačuhová ◽  
M. Uhrinčať ◽  
L. Hasoňová ◽  
...  
Keyword(s):  
Milk Yield ◽  
Somatic Cell Count ◽  
Peak Flow ◽  
Milk Composition ◽  
Latency Time ◽  
Peak Flow Rate ◽  
Order Of Entry ◽  
Milk Samples ◽  
Total Milk ◽  
The Individual

The aim of the present investigation was to evaluate how the order in which the ewes in a milking group enter the milking parlour affects their milkability and milk composition. Therefore, the order of entry into the milking parlour was evaluated in ewes of one flock (n = 353) during six evening milkings. In all, the sheep were milked in 15 milking groups. The sheep entering the milking parlour in the first milking group achieved 15 points and the sheep of each next milking group one point less, i.e. the sheep of the last group achieved 1 point. In the analysis, only the ewes with the highest and the lowest average number of points were included and assigned to the first (FG, n = 19) and the last group (LG, n = 29), respectively. After the last milking, the individual milk samples were collected from the jar to analyze the composition and somatic cell count. Machine milk yield in 30 s (0.15 ± 0.09 and 0.11 ± 0.05 l) and 60 s (0.26 ± 0.16 and 0.19 ± 0.10 l), peak flow rate (1.04 ± 0.39 and 0.77 ± 0.29 l/min), and latency time (14 ± 3 and 20 ± 13 s) significantly differed (P < 0.05) between FG and LG, respectively. Total milk yield (0.41 ± 0.17 and 0.35 ± 0.14 l) and machine milk yield (0.27 ± 0.15 and 0.22 ± 0.10 l) tended to be higher (P = 0.05 and P = 0.09) in FG than in LG, respectively. No significant differences were observed in milk composition between FG and LG. It seems that ewes which enter the milking parlour in early milking groups have better parameters of milkability than those milked in later groups.

Lactation and Energetics of Growth in the Brush-Tailed Bettong, Bettongia-Penicillata (Marsupialia, Potoroidae) in Captivity

1994 ◽  
Vol 42 (3) ◽  
pp. 267 ◽  
Author(s):  
JC Merchant ◽  
JA Libke ◽  
MJ Smith
Keyword(s):  
Maximum Size ◽  
Mammary Glands ◽  
Milk Composition ◽  
Solid Food ◽  
Body Tissue ◽  
Milk Consumption ◽  
Milk Samples ◽  
Bettongia Penicillata ◽  
In Captivity ◽  
Consumption Rates

Milk samples were collected from 20 captive bettongs at various times during the 140 days of lactation to determine milk composition. Milk consumption rates of young were measured, using Na-22 turnover, up to the time of their permanent emergence from the pouch and before solid food was consumed. Milk composition followed the general patterns of change observed previously in other marsupials and included the characteristic drop in carbohydrate concentrations associated with permanent exit from the pouch. Milk consumption rates increased from about 1.0 mL day(-1) at 35 days to about 23 mL day(-1) at 90 days, just before permanent emergence from the pouch. Increases in milk consumption were paralleled by changes in the size of the mammary glands, which reached their maximum size at about 100-110 days. The efficiency with which young used milk to accumulate body tissue ranged from 0.39 to 0.51 g mL(-1). Milk energy and protein were converted to body tissue with efficiencies of 21.3-29 6% and 70.1-93 5% respectively.

scholarly journals Effect of the stage of lactation on milk composition, its properties and the quality of rennet curdling in East Friesian ewes

2008 ◽  
Vol 53 (No. 2) ◽  
pp. 55-63 ◽  
Author(s):  
J. Kuchtík ◽  
K. Šustová ◽  
T. Urban ◽  
D. Zapletal
Keyword(s):  
Titratable Acidity ◽  
Milk Composition ◽  
The Other ◽  
Phenotypic Correlation ◽  
Clotting Time ◽  
Milk Samples ◽  
Phenotypic Correlations ◽  
Stage Of Lactation ◽  
Sheep Farm

The evaluation of the effect of the stage of lactation on milk composition, its properties and the quality of rennet curdling was carried out over the period of three successive years using milk samples (<I>n</I> = 162) obtained from a total of 27 ewes of the East Friesian (EF) breed, reared on a small sheep farm in Juřinka in the region of Wallachia. The stage of lactation had a highly significant effect on the contents of all milk components. However, only the contents of total solids (TS), solids non-fat (SNF), fat (F), protein (P) and casein (CN) gradually increased with the advancement of lactation. The stage of lactation also had a highly significant effect both on all the properties of milk and the rennet curdling quality (RCQ). All phenotypic correlations between the particular contents of TS, SNF, F, P, CN and urea nitrogen (UN) were positive and high (<I>P</I> ≤ 0.001). On the other hand, all phenotypic correlations between milk yield and particular contents of TS, SNF, F, P, CN and UN were negative and high (<I>P</I> ≤ 0.001). The majority of phenotypic correlations between rennet clotting time (RCT) and the other particular parameters was insignificant. However, the phenotypic correlations between lactose (L) and RCT and between pH and RCT were positive and high (<I>P</I> ≤ 0.001) whereas the phenotypic correlation between titratable acidity (TA) and RCT was negative and high (<I>P</I> ≤ 0.001). The majority of phenotypic correlations between the rennet curdling quality (RCQ) and the other particular parameters was insignificant. Nevertheless, the phenotypic correlations between pH and RCQ and between RCT and RCQ were positive and high (<I>P</I> ≤ 0.001) whereas the phenotypic correlation between TA and RCQ was negative and high (<I>P</I> ≤ 0.001).

Dependence between ‘heat diarrhoea’ traits in foals and mares’ milk composition

2006 ◽  
Vol 35 ◽  
pp. 287-291
Author(s):  
M. Pieszka ◽  
M. Kulisa
Keyword(s):  
Growth And Development ◽  
Milk Composition ◽  
Milk Samples ◽  
Main Components ◽  
The Relationship

Growth and development of foals is highly influenced by different factors during the period they spend with their mothers. One of the most interesting and not really recognised phenomena is first foals’ diarrhoea called ‘heat diarrhoea’ or ‘heat scours’. This diarrhoea occurs during first 2-3 weeks after birth in most of horse breeds (Deskur et al. 1978, Gustafsson 1998). Until today there is no exact explanation of this phenomenon – most vets describe it as a pathological infection due to viruses or bacteria, parasitologists explain it as an early invasion of some nematodes, physiologists describe it as a result of changes in intestines and breeders clarify it as a result of changing in mare's milk composition related to her heat. The objective of this study was to investigate the relationship between first diarrhoea occurrence, its duration and intensity and mares’ milk composition.The research was carried out on 30 Arabian mares and their foals. Milk samples were collected to evaluate the main components and macroelements.

scholarly journals A Comparison of Changes in the Fatty Acid Profile of Human Milk of Spanish Lactating Women during the First Month of Lactation Using Gas Chromatography-Mass Spectrometry. A Comparison with Infant Formulas

Nutrients ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 3055 ◽  
Author(s):  
Silvia Sánchez-Hernández ◽  
Adelaida Esteban-Muñoz ◽  
Rafael Giménez-Martínez ◽  
María José Aguilar-Cordero ◽  
Beatriz Miralles-Buraglia ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Fatty Acid ◽  
Human Milk ◽  
Milk Composition ◽  
Gas Chromatography Mass Spectrometry ◽  
Infant Formulas ◽  
Mature Milk ◽  
Lactating Women ◽  
Milk Samples

Breastfeeding is the ideal way to provide infants with the nutrients they need for healthy growth and development. Milk composition changes throughout lactation, and fat is one of the most variable nutrients in human milk. The aim of this study was to determine the main differences between the fatty acid (FA) profile of human milk samples (colostrum, transitional, and mature milk group) and infant formulas. Human milk samples were provided by lactating women from Granada. Moreover, different commercial infant formulas were analyzed. FAs were determined using gas chromatography coupled with mass spectrometry. According to the results, oleic acid was the predominant monounsaturated fatty acid (41.93% in human milk and 43.53% in infant formulas), while palmitic acid was the most representative saturated fatty acid (20.88% in human milk and 23.09% in infant formulas). Significant differences were found between human milk groups and infant formulas, mainly in long-chain polyunsaturated FAs (LC-PUFAs). The content of araquidonic acid (AA) and docoxahexaenoic acid (DHA) was higher in human milk (0.51% and 0.39%, respectively) than in infant formulas (0.31% and 0.22%, respectively). Linoleic acid (LA) percentage (15.31%) in infant formulas was similar to that found in human milk (14.6%). However, α-linolenic acid (ALA) values were also much higher in infant formulas than in human milk (1.64% and 0.42%, respectively).

MRD Monitoring in AML Patients with MLL-MLLT4 by Quantification of Fusion Gene Transcripts and Genomic Chromosomal Breakpoint Sequences

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4888-4888
Author(s):  
Grigory Tsaur ◽  
Anna Ivanova ◽  
Olga Plekhanova ◽  
Tatyana Riger ◽  
Yulia Yakovleva ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Fusion Gene ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Exon 2 ◽  
Standard Curve ◽  
Healthy Donors ◽  
Intron 1 ◽  
Exon 9 ◽  
Chromosomal Breakpoint

Abstract Statement. The MLL-MLLT4 (former MLL-AF6) fusion gene (FG) is relatively rare genetic abnormality, predominantly found in AML. It averages 3–5% among other MLL rearrangements. Here we present data of MRD monitoring in 2 patients with AML carrying an MLL-MLLT4 rearrangement by 2 approaches: using FG transcript at RNA/cDNA level in comparison with FG at genomic DNA level. Materials and methods. Patients (pts) were diagnosed according French-American-Britain (FAB) classification. Initial diagnostics included cytomorphology, immunophenotyping of blast cells, cytogenetics, FISH and reverse transcriptase PCR (RT-PCR). RT-PCR products were directly sequenced afterwards. In both cases identification of genomic chromosomal breakpoint sequences within MLL and MLLT4 genes was done by long-distance inverse PCR (LDI-PCR). MRD quantification in genomic DNA was performed using patient-specific primers and probes by real-time quantitative PCR (RQ-PCR). 500 ng of DNA was used per reaction. Standard curve was received by serial 10-fold dilutions of pts’ DNA into the DNA isolated from pooled lymphocytes of ten healthy donors. b-actin was used as DNA quality and quantity control. Detection of FG transcript kinetics during treatment was performed by RQ-PCR according to “Europe Against Cancer” recommendations for normalization by using control gene ABL (Gabert J. et al Leukemia, 2003, 17) and for using 10-fold dilutions of plasmids carrying MLL-MLLT4 fragment as source of standard curve. MRD value for cDNA targets were estimated as previously described (Beillard E. et al Leukemia, 2003, 17). Each sample was run in triplicates. According to the treatment design, time-points for MRD estimation were scheduled before each block of treatment. Totally 5 samples were evaluated in each patient (initial and 4 follow-ups). FLT3-ITD status was estimated at the time of diagnosis. Informed consent was obtained in both cases. Patients’ characteristics, treatment and clinical outcome Case # 1 Case #2 Age 58 13 Sex Male Male Initial WBC*106/ml 5.5 94.9 Immunophenotype CD34+CD117+HLA-DR+ CD11c+CD13+CD33+CD65+ CD34+CD117+CD13+ CD33+CD45+MPO+ Cytogenetics 46, XY, del(5)(q?), der(5)t(5;6;11) (q22;q15q27;q23), der(6)t(5;6) (q22;q15), del der(11) 46, XY, t(6;11)(q27;q23) FISH with LSI MLL MLL deletion MLL split RT-PCR MLL-MLLT4 positive MLL-MLLT4 positive MLL-MLLT4 FG transcript exon 9-exon 2 exon 9-exon 2 Localization of genomic chromosomal breakpoint within MLL and MLLT4 intron 9-intron 1 intron 9-intron 1 FLT3-ITD Negative Negative Induction treatment 7+3 AIE Consolidation therapy 2× HAM 2× HDAC 1× HAM 1× FLAG 1× HAE Maintenance − + Duration of therapy, months 8 7 Achievement of CR + + OS, months 8 7 EFS, months 6 5 Current status Alive in CR Alive in CR Results. Despite of achievement of CR, MLL-MLLT4 FG transcripts were detected in every sample tested after induction and consolidation chemotherapy by RQ-PCR. MRD value in case #1 in cDNA was fluctuated significantly within 2 log. Although in case #2 there was successive reduction from 260% at the beginning of treatment till 0.7% before maintenance therapy (after HAE block). Limited dilution series of a MLL-MLLT4-positive RNA into RNA of ten healthy donors showed a sensitivity limitation of 1E–05. For quantification of genomic chromosomal breakpoint sequences b-actin was amplified in each well. Deviation between Ct values of b-actin in different wells did not exceed ±2.0. In case # 1 the standard curve of the RQ-PCR assay for MLL-MLLT4 FG had slope of −3.19. Correlation coefficient was 0.987. Quantitative range of this assay was 1E–04 and sensitivity 1E–5. It was also observed a considerable variation of MRD levels in genomic DNA during treatment, like it was observed in MRD monitoring by FG transcripts. Fluctuations run up to 2.5 log. In case #2 the standard curve of the RQ-PCR assay for MLL-MLLT4 had a slope of −3.63 with correlation coefficient 0.992. Quantitative range of this assay was reached 1E–04 with sensitivity 1E–05. MRD level in this patient constantly decreased. Conclusions. The same tendency has been shown in each patient: fluctuations of MRD levels (2.5 log in case #1) and successive reduction (case #2). Results received at RNA/cDNA level and in genomic DNA cannot substitute each other, but they can be used as additives. It has been demonstrated that quantification of MLL-MLLT4 FG in genomic DNA is precise and suitable for MRD monitoring.

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