Sequence-specific Oligonucleotide Probes Research Articles

High frequency of HLA-A∗0103 allele in a Somali population

We report the existence of class I HLA allele A∗0103 in an ethnic group (Somali) where this allele has not been reported. This allele was discovered in a study to examine the relationship between HLA alleles and humoral antibody response to measles vaccine among recent immigrants from Somalia to Olmsted County, Minnesota. We initially used polymerase chain reaction–sequence-specific primers (PCR-SSP) to carry out HLA class I typing. Based on PCR-SSP, 55 subjects were assigned the allele HLA-A∗0101. Following direct DNA sequencing of the PCR products, 37 of the 55 subjects (67.3%) that were initially assigned the A∗0101 allele were found to actually be A∗0103. Our data are significant because it demonstrates that many of the previously typed A∗0101 individuals are actually A∗0103 as the SSP or sequence-specific oligonucleotide probes method cannot distinguish between the two alleles. Lastly, this is the first identification of this allele in the homozygous state.

Quantitative plasmid mixture analysis using the fluorogenic 5'-nuclease polymerase chain reaction assay.

The fluorogenic 5'-nuclease polymerase chain reaction (PCR) assay has been shown to be useful for quantifying a given DNA target in a sample. Here we show how an existing PCR protocol can be amended for quantification by incorporating distinctive dual-labeled, sequence-specific oligonucleotide probes and resulting in a two- to threefold broader and more reliable dynamic range than that of conventional end-point analysis of PCR products. Moreover, we show a multiplex situation in which two targets, one normal and one mutated, can be amplified and quantified simultaneously and in the same reaction tube. Use of this novel approach for quantitative PCR applications eliminates the need for post-PCR processing and has clinical- and research-based diagnostic applications, particularly for measuring levels of mutations in a mixture.

Extent and Distribution of Linkage Disequilibrium in Three Genomic Regions

The positional cloning of genes underlying common complex diseases relies on the identification of linkage disequilibrium (LD) between genetic markers and disease. We have examined 127 polymorphisms in three genomic regions in a sample of 575 chromosomes from unrelated individuals of British ancestry. To establish phase, 800 individuals were genotyped in 160 families. The fine structure of LD was found to be highly irregular. Forty-five percent of the variation in disequilibrium measures could be explained by physical distance. Additional factors, such as allele frequency, type of polymorphism, and genomic location, explained <5% of the variation. Nevertheless, disequilibrium was occasionally detectable at 500 kb and was present for over one-half of marker pairs separated by <50 kb. Although these findings are encouraging for the prospects of a genomewide LD map, they suggest caution in interpreting localization due to allelic association.

Further diversity at HLA-A and -B loci identified in Afro-Caribbean potential bone marrow donors.

Two novel HLA-A and three novel HLA-B alleles were identified within a group of Afro-Caribbean individuals who were recruited as potential donors for the Anthony Nolan Bone Marrow Trust Register. HLA typing was performed on DNA extracted from peripheral blood mononuclear cells using sequence-specific oligonucleotide (SSO) probes for HLA-A and -B loci. Eight individuals analysed exhibited hybridisation patterns for which a type could not be assigned. DNA from these individuals was further typed by two methodologies: direct sequencing of PCR products and reference strand conformation analysis (RSCA). The direct sequencing results allowed the identification of new alleles but did not allow confirmation of the cis/trans orientation of the new sequence motifs identified. RSCA analysis confirmed the results obtained by SSO and direct sequencing and in addition confirmed the cis/trans orientation of the new sequences. One individual possesses a new A*30 allele--A*3008 and two individuals possess an identical new A*74 allele--A*7404. The three novel HLA-B alleles were identified in three individuals: B*0812, B*1554 and B*4503 respectively. For the remaining two samples, A*2612 was identified. At present Caucasoid individuals, and therefore Caucasoid phenotypes, are predominantly represented on the various different volunteer bone marrow donor registries. The examples presented here highlight the potential for identification of further polymorphisms within the HLA system as more individuals from the much-needed ethnic minorities are recruited onto bone marrow donor registers.

Diversity and Heterogeneity in Mitochondrial DNA of North American Populations

Variation in the mitochondrial DNA (mtDNA) control region as detected by sequence-specific oligonucleotide (SSO) probes is described for 2282 individuals from African-American, European-American, and Hispanic subpopulations from five broadly defined regions of North America (Northeast, Southeast, Central, Northwest, Southwest). Population diversity estimates were uniformly high for all subpopulations and for each major ethnic group. Only the Pennsylvania Hispanic group was remarkable with respect to its mitochondrial DNA types, having both six low frequency population specific types (ranging from 1.2-8.6%) and three high frequency shared types (10-20% each). There was no statistically significant subpopulation heterogeneity present within any of the three major groups at either the subpopulation level or the regional level (p > 0.01). However, statistically significant heterogeneity was measured when comparing the three major groups to each other, with the variance component attributable to this large division accounting for 18.60% of the total variance (p < 0.001). Overall mtDNA is a satisfactory forensic typing locus within broadly defined African-American, European-American, and Hispanic groups from North America, based on the high diversity estimates and absence of heterogeneity, as characterized by SSO typing.

Immunogenetics of HLA class II in Israeli Ashkenazi Jewish, Israeli non-Ashkenazi Jewish, and in Israeli Arab IDDM patients

The distribution of HLA class II alleles and genotypes in IDDM patients was examined in the three main Israeli ethnic groups: Ashkenazi Jews, non-Ashkenazi Jews, and Arabs. Molecular sequence specific oligonucleotide probe analysis was performed for DRB1, DQA1, and DQB1 genes. The DRB1∗03011, DQA1∗05 DQB1∗02/DRB1∗0402, DQA1∗03, DQB1∗0302 genotype was found to be the main susceptibility genotype in all three groups, with differences in the degree of association. In addition to DRB1∗0402 (more frequent among Ashkenazi Jews), DRB1∗0405, another subtype of DRB1∗04, was found to be more prevalent among non-Ashkenazi Jews and Arabs. Many alleles were found to be negatively associated with insulin dependent diabetes mellitus (IDDM). This could be a result of the high frequency of susceptible alleles, or of linkage disequilibrium to a primary negatively associated allele. The strongest negative association was observed for DQB1∗0301 in all three ethnic groups. The alleles DRB1∗1401, DRB1∗1501, DQB1∗05031, DQB1∗0602, and DQB1∗0609 were not detected in any of the 202 IDDM patients, and are probably either strongly protective or in linkage with such alleles. Despite the differences found between the three ethnic groups, an overall analysis shows that the DRB1∗04 alleles that account for susceptibility to IDDM in the Israeli population (DRB1∗0402 and ∗0405) are the same as those responsible for susceptibility to IDDM in a number of other Mediterranean populations. In contrast, the susceptible allele in most Caucasian populations is DRB1∗0401. It is noteworthy that the susceptible alleles DRB1∗0402/05 for Mediterranean and DRB1∗0401 for Caucasian populations are also frequent in the respective healthy populations. These findings support the results obtained in other studies, which point to a genetic relationship between the Israeli and Mediterranean populations.

HLA class II typing of whole genome amplified mouth swab DNA.

Postal collection of mouth swabs provides a cheap and convenient means of DNA sampling but hitherto has not provided sufficient genetic material for HLA typing by polymerase chain reaction using sequence-specific primers (PCR-SSP). This study examined the feasibility of collecting mouth swabs from a test population by post, amplifying the DNA by whole genome amplification and genotyping for selected HLA class II alleles. We optimised a strategy for whole genome amplification or primer extension preamplification using a random 15 base pair primer which resulted in a 1,000-fold increase in DNA template. The amplified DNA was of sufficient quality for analysis of selected HLA Class II alleles by PCR-SSP and PCR using sequence-specific oligonucleotide probes. To test the reliability of our data, blood DNA from 30 individuals in 10 families, previously tested for all DRB1 alleles in a routine diagnostic laboratory, was then tested in our laboratory for DRB1 *03 and *04 following whole genome amplification. Further whole genome amplified product from another 10 families was tested for DRB1 *03, *04 in our laboratory and then tested for all DRB1 alleles in a routine diagnostic laboratory. One repeat typing was required to achieve 100% concordance between laboratories. Amplification of whole genome amplified DNA by PCR-SSP was then extended successfully to low-resolution HLA DRB1, DQA1, DQB1 and DPB1 typing. Mouth swab collection by post, followed by whole genome amplification of DNA provides an effective strategy for genetic analysis of large cohorts. We have optimised conditions for HLA class II typing on whole genome amplified DNA collected by mouth swab, but this method could potentially be applied to low concentrations of DNA from other sources.

HLA-B*15 diversity in the Korean population.

Alleles in the HLA-B*15 group encode molecules belonging to several serologic subgroups, B15 (B62, B63, B75, B76, B77) and B70 (B71, B72), representing many of the most problematic types to assign in routine clinical typing laboratories due to their serologic cross-reactivity resulting from structural similarity. More than 25% of Koreans express HLA-B molecules encoded by the HLA-B*15 alleles. To further characterize HLA-B*15 in this population, B*15-specific polymerase chain reaction (PCR) and sequence-specific oligonucleotide probe (SSOP) hybridization analysis using 39 digoxigenin-labeled probes were applied to DNA samples obtained from 237 B15/B70 serologically positive unrelated individuals. Nine B*15 alleles were identified. B*1501 was the most frequent allele (64.8%) followed by B*1511 (14.1%), B*1507 (8.6%), and B*1518 (5.5%) comprising more than 90% of B*15-positive samples. B62 molecules encoded by 4 of the identified alleles (B*1501, B*1507, B*1525, and B*1527) could not be discriminated by serologic reaction patterns. Among the fifteen B15/B70 apparent homozygotes, eight were heterozygotes carrying two different B*15 alleles. Several B*15 alleles exhibited strong associations with specific Cw, DRB1, and A allelic types (e.g., B*1507-Cw3 (22/22); B*1507-DRB1*04 (21/22), B*1507-A24 (17/22)). The data obtained in this study confirmed B*15 diversity in the study population and will be useful in hematopoietic stem cell donor searches as well as in determining the supplementary DNA typing strategy for B15/B70-positive samples in this population.

Detection of Sequence Variation in the HVII Region of the Human Mitochondrial Genome in 689 Individuals Using Immobilized Sequence-Specific Oligonucleotide Probes

We have developed a rapid, immobilized probe-based assay for the detection of sequence variation in the hypervariable segment II (HVII) of the mitochondrial DNA (mtDNA) control region. Using a panel of 17 sequence-specific oligonucleotide (SSO) probes immobilized on nylon membrane strips, we typed 689 individuals from four population groups. The genetic diversity value for each population was calculated from the frequency data, and the frequencies of distinct "mitotypes" in each group were determined. We performed DNA sequence analysis of 129 samples to characterize the sequences associated with "blanks" (absence of probe signals) and weak probe signals. Out of 689 samples, we observed five heteroplasmic samples (excluding the variable C-stretch beginning at position 303) using the immobilized SSO probe panel. The SSO probe strips were used for the analysis of shed hairs and bloodstains from several criminal cases in Sweden, one of which is described here. We conclude that this mtDNA typing system is useful for human identification and significantly decreases casework turnaround time.

Diversity is demonstrated in class I HLA-A and HLA-B alleles in Cameroon, Africa: description of HLA-A*03012, *2612, *3006 and HLA-B*1403, *4016, *4703.

To examine the genetic diversity in west Africa, class I HLA-A and HLA-B alleles of 92 unrelated individuals from two areas in the Cameroon, the capital Yaoundé and the village of Etoa, were identified by direct automated DNA sequencing of exons 2 and 3 of the HLA-B locus alleles and sequence-specific oligonucleotide probe (SSOP) and/or sequencing of the HLA-A locus alleles. HLA-A*2301 (18.7%), A*2902 (10.4%), B*5301 (10.9%), and B*5802 (10.9%) were the most frequently detected alleles, present in at least 10% of the population. A total of 30 HLA-A locus and 33 HLA-B locus alleles, including six novel alleles, were detected. The novel alleles were HLA-A*03012, A*2612, A*3006 and HLA-B*1403, B*4016, and B*4703. HLA-B*4703 contains a novel amino acid sequence that is a combination of the first 5 amino acids of the Bw6 epitope and the last 2 residues of the Bw4 epitope. The addition of 6 alleles to the ever-expanding number of known class I HLA alleles supports our hypothesis that extensive genetic diversity, including previously undescribed alleles, would be observed in this African population. In the Yaoundé population, the allele frequency distribution at the HLA-A locus is consistent with distributions indicative of balancing selection. Extensive HLA-A-B haplotypes were observed in this population suggesting that only a fraction of the Cameroon HLA-A-B haplotype diversity has been observed.

Development of a PCR-SSOP approach capable of defining the natural killer cell inhibitory receptor (KIR) gene sequence repertoires.

A molecular typing method based on polymerase chain reaction (PCR) amplification of three different target domains (immunoglobulin domains 1 and 3, and the transmembrane-cytoplasmic domain), followed by hybridisation with 26 digoxigenin-labelled sequence-specific oligonucleotide probes (SSOP) has been established for the polymorphic killer inhibitory receptor (KIR) genes. In addition to identifying the 12 KIR subfamilies, our PCR-SSOP typing approach could also distinguish the putative alleles, NKB1 and NKAT3, that comprise the KIR3DL1 subfamily. Ninety unrelated blood donors and 13 families (52 individuals), including both parents, were subjected to our KIR PCR-SSOP typing approach. All 12 KIR subfamilies, including a 2DS5 variant sequence, were present in the 90 individuals and displayed varied phenotype frequencies: 2DL1 (0.96), 2DL2 (0.31), 2DL3 (0.95), 2DS1 (0.56) 2DS2 (0.51), 2DS3 (0.27), 2DS4 (0.96), 2DS5v (0.35), 3DS1 (0.47), 3DL1 (0.96), 3DL2 (1.0) and 2DL4 (1.0). A total of 23 different KIR phenotypes were defined in this study, and 10 of these were only found on one occasion in one individual, indicating considerable diversity in the KIR phenotype profiles within the Irish population. Most individuals (93%) possessed the complement of inhibitory KIR specificities for the three well-defined HLA-B and -C ligands. An unusual probe pattern for 3DS1 was observed in 3 individuals indicating a variant 3DS1 gene sequence with changes at nucleotide positions 1185-1186, within the cytoplasmic domain. Sequencing analysis revealed a new single nucleotide polymorphism in exon 3 of 3DL1 NKB1(195, G-A) and a 22-bp deletion polymorphism in exon 5 of 2DS4 (nucleotides 777-798 deleted). A number of strong KIR associations were observed, namely 2DL1 with 2DL3, 2DS4 with 3DL1, 2DL2 with 2DS1/2DS2/2DS3, 2DS1 with 2DS3/2DS5v/3DS1, 2DS2 with 2DS3 and 2DS5v with 3DS1. Analysis of the KIR segregation observed in the 13 families confirmed these strong associations and permitted the definition of a number of partial KIR haplotypes, e.g. 2DL2-2DS1-2DS2-2DS3-3DL1. The segregation analysis concluded that at least 3 distinct gene loci encode 2DL1-4 and at least 4 gene loci encode the non-inhibitory KIR2DS1-2DS5. In the case of 3DL1-2 and 3DS1, our data suggests 3 gene loci, one for each subfamily.

HLA class I alleles associated with susceptibility or resistance to human immunodeficiency virus type 1 infection among a population in Chaco Province, Argentina.

Host genetic factors, such as HLA alleles, are important in human immunodeficiency virus (HIV) infection and its progression to AIDS. HLA class I gene products are involved in peptide presentation, and each allele is responsible for presenting a different set of peptides to cytotoxic T lymphocytes. The increase or decrease in the frequency of certain alleles in HIV-1-positive versus control subjects would suggest that alleles play a role in susceptibility to or protection against this viral infection. In the present study, 56 HIV-1-positive patients in Chaco Province, Argentina, were typed for the HLA-A and HLA-B loci, using polymerase chain reaction and sequence-specific oligonucleotide probes. The frequency of alleles A*24, B*18, and B*39 was increased in HIV-1-positive subjects, suggesting that these alleles play a role in susceptibility to HIV-1 infection. Alleles B*44 and B*55 were not found in HIV-1-positive subjects, suggesting that they have a protective effect against the disease. The P values of the alleles B*39 and B*44 were statistically significant (P<.05).

Renal failure after clinical heart transplantation is associated with the TGF-β1 codon 10 gene polymorphism

Background: To determine whether genetic factors are involved in the development of renal dysfunction due to cyclosporine nephrotoxicity, we analyzed 2 polymorphisms in the signal sequence of the transforming growth factor (TGF)–β1 gene; codon 10 (Leu 10 → Pro) and codon 25 (Arg 25 → Pro). Method Using sequence specific oligonucleotide probing, we analyzed both TGF-β1 gene polymorphisms in cardiac allograft recipients ( n = 168) who survived at least 1 year with minimal follow-up of 7 years. Patients received cyclosporine and steroids as maintenance immunosuppressive therapy. Renal dysfunction was defined as a serum creatinine ≥ 250 μmol/liter. Results Renal dysfunction was observed in 2% (3/168) of the patients at 1 year, in 7% (11/160) at 3 years, in 12% (18/152) at 5 years, and in 20% (26/131) at 7 years post-transplantation. The genotypic distributions for TGF-β1 codon 10 were 7% Pro/Pro, 61% Pro/Leu, and 32% Leu/Leu, and for codon 25 these percentages were 1% Pro/Pro, 12% Pro/Arg, and 87% Arg/Arg. We found an association between the TGF-β1 genotype encoding proline at codon 10 and renal dysfunction. At 7 years post-transplantation, 26% (23/89) of the patients with the heterozygous Pro/Leu or homozygous Pro/Pro genotype had renal dysfunction vs only 7% (3/42) of the patients with the homozygous Leu/Leu genotype ( p = 0.017). For the TGF-β1 codon 25 genotypes, we found no association between TGF-β1 genotypes and renal dysfunction. Conclusion Our data support the hypothesis that TGF-β1 is involved in the process leading to renal insufficiency in cyclosporine-treated cardiac allograft recipients. In these patients the presence of TGF-β1 Pro 10 might be a risk factor.

HLA-DRB1 and DQB1 polymorphisms in Southern France and genetic relationships with other Mediterranean populations

This study presents the results of HLA-DRB1 and DQB1 sequence-specific oligonucleotide probe (SSOP) typing for a population sample of 181 individuals originating from southern France. On the basis of allele and haplotype frequencies, we compared our population with others from the Mediterranean area. Allele frequencies are comparable to those found in other western European populations (France, Portugal, Spain) and indicate neighboring exchanges. The haplotype frequencies showed relationships with North Africans and Jewish populations, as well as the common origin of Moroccan and Lebanese Jews. Therefore, allele frequencies seem to be more able to show recent exchanges while haplotype frequencies might show ancestral relationships. These results may serve as references for future studies of HLA and disease in southern France.

Identification of a novel allele, DRB1*1340, in two Brazilian individuals.

We identified a novel HLA-DRB1 allele, named DRB1*1340 by the WHO HLA Nomenclature Committee, in two Brazilian individuals. Typing by polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) showed a DRB1*13 allele with an unusual hybridization pattern. DNA sequencing of both strands and comparison of the sequence with previously described DRB1 alleles revealed that the most similar allele is DRB1*1301, from which DRB1*1340 differs by a single nucleotide (T-->A) in exon 2, at position 127, codon 47 (Phe-->Tyr). The sequence received accession number AJ237964 from the EMBL database.

Going back to the roots: effective utilisation of HLA typing information for bone marrow registries requires full knowledge of the DNA sequences of the oligonucleotide reagents used in the testing

Information obtained by DNA-based HLA typing assays is more detailed and of higher quality than that obtained by conventional serological techniques. Nevertheless, it is common for data acquired in this way to be presented in the more familiar serological format. In many cases this representation can lead to significant loss of information, which may only become apparent at a later time, with the discovery of novel allele sequences. DNA-based typing methods, such as sequence-specific oligonucleotide probing (SSOP) or sequence-specific priming (SSP) generate fragmentary sequence data which is information rich. An alternative to assigning allele names to these fragments is to simply store the sequence data itself without interpretation. Bone marrow donor repositories can then be searched directly with sequence information, which though complex is more complete, rather than searching by derivative allele names.

Sequence-specific oligonucleotide probing for MICB alleles reveals associations with MICA and HLA-B.

Sequence-specific oligonucleotide probing for MICB alleles reveals associations with MICA and HLA-B.

DRB3 alleles with variations in the annealing sites of commonly used amplification primers.

New HLA alleles are often identified initially from observing uncommon patterns found in low-resolution typing performed via polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP). Recently, the HLA-DR oligotyping analysis of two Caucasian, one Caucasian/American Indian and two African American individuals resulted in the identification of three novel DRB3 alleles. Using DRB-specific primer sets commonly employed in amplification-based typing, all four individuals were originally characterized as DRB3 negative. Direct sequencing identified DRB3*0104 (variation at codon 8, TCG instead of TTG), and DRB3*0101202 (variation at intron (-13), G instead of C). One individual appeared to carry a DR52-associated DRB1 allele without an associated DRB3 allele. Lack of conservation at the junction of intron 1 and exon 2 of the DRB3 gene suggests that commonly used DRB-specific primer sets may need to be modified.

A polymorphism of the 5' flanking region of tumour necrosis factor α gene is associated with thyroid-associated ophthalmopathy in Japanese.

We have studied the polymorphism of the 5' flanking region of the tumour necrosis factor (TNF)-α gene in order to better understand the genetic background of autoimmune thyroid disorders and thyroid-associated ophthalmopathy. We studied the polymorphism of the 5' flanking region of the TNF-α gene at positions - 1031 (T to C change, termed as - 1031C), - 863 (C to A, - 863 A), - 857 (C to T, - 857T), - 308 (G to A, - 308 A) and - 238 (G to A, - 238 A) in Japanese patients with Graves' disease [n = 173, 62 of whom had associated ophthalmopathy (American Thyroid Association (ATA) class III or greater)] and healthy control subjects (n = 575), using a polymerase chain reaction sequence-specific oligonucleotide probe method. The allele frequency of - 857T in the Graves' disease patients (22.5% vs. 17.7%, OR = 1.35, P = 0.045, corrected P = 0.23) was slightly greater than in the Japanese healthy subjects, respectively. However, the difference was not statistically significant. In Graves' disease patients with evident ophthalmopathy (ATA class III or greater), the allele frequencies of - 1031C and - 863 A were significantly greater than those with no or mild ophthalmopathy (ATA class 0-II) (31.5% vs. 13.5%, OR =2.94, P < 0.0001, corrected P < 0.0005; 23.4% vs. 11.7%, OR =2.30, P = 0.0044, corrected P = 0.022, respectively) and in control subjects. The strength of the association of the polymorphism - 1031C increased with the severity of ophthalmopathy, with odds ratios of 2.36 for ATA class III, and 5.43 for ATA class IV-VI, respectively, compared with Graves' disease with no or mild ophthalmopathy (ATA class 0-II). Although the phenotype frequency of DRB1*0901 was not different among Graves' disease patients with or without ophthalmopathy and control subjects, the phenotype frequency of DRB1*0901(-)/-1031C(+) was significantly increased in Graves' disease patients with ophthalmopathy compared to those with no or mild ophthalmopathy (OR = 4.91, P = 0.0005) or control subjects (OR = 4.59, P < 0.0001). These results suggest that the - 1031C or - 863 A alleles, or a gene in linkage disequilibrium with the TNF-α gene, predispose to the development of ophthalmopathy in Japanese patients with Graves' disease.

Kappa-casein and beta-Casein alleles in crossbred and Zebu cattle from India using polymerase chain reaction and sequence-specific oligonucleotide probes.

Caseins constitute the main part of milk proteins. The genes for the four major types of casein (CN), αs1-CN, αs2-CN, β-CN and κ-CN, reside on &lt; 220 kb of the DNA on bovine chromosome 6 (Threadgill &amp; Womack, 1990). Two casein genes (Cn), β-Cn and κ-Cn, have been found to be associated with differences in milk production, cheese yield, protein content, quality and fat yield (Ng-Kwai-Hang et al. 1984). Lin et al. (1986) have estimated that the combined contributions of αs1-Cn, β-Cn, κ-Cn and β-lactoglobulin loci accounted for 8·9% of the total phenotypic variance in milk, 8·6% of that in proteins and 5% of that in fat yield. Six alleles of κ-Cn gene have so far been identified: κ-CnA, κ-CnB, κ-CnC, κ-CnE, κ-CnF and κ-CnG. The κ-CN protein is 169 amino acids long with varying regions at codons 136 and 148. The A variant has threonine (ACC) at codon 136 and aspartic acid (GAT) at codon 148, whereas the B variant has isoleucine (ATC) and alanine (GCT) at codons 136 and 148 respectively (Mercier et al. 1973). Milk containing κ-CN B was reported to yield more cheese than that with κ-CN A because it contains more casein and has lower fat loss. Several studies have shown that cows with the κ-CnBB genotype produced milk with a higher protein content than cows with κ-CnAA (Ng-Kwai-Hang et al. 1984; Ron et al. 1994).