| First Author | Jones J | Year | 1993 |
| Journal | Mouse Genome | Volume | 91 |
| Issue | 1 | Pages | 124-26 |
| Mgi Jnum | J:4239 | Mgi Id | MGI:52735 |
| Citation | Jones J, et al. (1993) Discrimination of mouse beta-globin haplotypes by allele specific amplification. Mouse Genome 91(1):124-26 |
| abstractText | Full text of Mouse Genome contribution: Discrimination of mouse Beta-globin haplotypes by Allele Specific Amplification. Janet Jones & Josephine Peters; MRC Radiobiology Unit, Chilton, Didcot, Oxon OX11 ORD. ABSTRACT The ability to discriminate the polypeptide chains determined by two commonly occurring mouse Beta-globin haplotypes, Hbbd and Hbbs, has been exploited in many mapping studies. The position of Hbb is so well known from extensive data that it is a chromosome 7 anchor. However, the current trend in haplotype analysis is to use DNA rather than protein base methodologies. Here we describe the use of Allele Specific Amplification (ASA) for the rapid determination of Beta-globin haplotype. Primers specific for the Hbb-b2d and Hbb-b2s alleles have been used to distinguish Hbb haplotypes and the results are concordant with those obtained by protein electrophoresis. Thus, the ability to detect variability at Hbb has been extended. INTRODUCTION Two Beta-globin haplotypes, Hbbd and Hbbs are common in adult laboratory mice. Hbbd determines two polypeptide chains that differ in 9 out of their 146 amino acid residues (Gilman, 1976). The predominant polypeptide chain, Betad major, is encoded by Hbb-b1d and the less abundant polypeptide chain, Betad minor, by the non allelic gene, Hbb-b2d. Two Beta-globin genes (Hbb-b1s and Hbb-b2s) are also expressed in the adult mice of the Hbbs haplotype. However, they determine polypeptide chains, Beta-single, with identical amino acid compositions (Popp 1973, Lewis et al., 1985). All four genes, Hbb-b1d, Hbb-b1s, Hbb-b2d and Hbb-b2s have been cloned and sequenced (Tiemeir et al, 1978; Konkel et al, 1979; Erhart et al, 1985) and have been shown to have a very similar gross structure (Fig 1). At the nucleotide level Hbb b1d and Hbb-b2d exhibit greater sequence divergence than their Hbbs counterparts over the regions bordered by their start and stop codons. The largest number of sequence differences occur between the two Hbb-b2 alleles, Hbb-b2d and Hbb-b2s (Erhart et al., 1985). The ability to detect genetic variability at the nucleotide level has been greatly enhanced bv the technique of allele specific amplification (Newton et al, 1989), which is sensitive enough to detect single nucleotide differences between closely related alleles in the mouse Beta-globin complex (Jones & Peters, 1991). With this technique Hbb-b1 can be discriminated from Hbb-b2 using primer sequence from the untranslated regions (Jones & Peters, 1991) but these primers will not distinguish alleles of either gene. Here we report the design of primers specific for the Hbb-b2s and Hbb-b2d alleles and their use in an allele specific amplification reaction for the rapid typing of Hbb haplotypes. Fig 1 - (Legend). Alignment of the allele specific Hbb-b2 primers to a DNA sequence that differs beween Hbb-bl, Hbb-b2d and Hbb-b2s. The sequence shown are taken from Erhart et al, 1985, only nucleotides that show differences in Hbb-b2 are shown. Nucleotide number 1 is at the cap site and the amplification primers [1a] and [1b] have 3' termini that align to nucleotide 15. A deliberate sequence alteration, indicated by a lower case letter, has been introduced close to the 3' end of the primers which will produce a mismatch with otherwise complementary DNA sequences. Primer 2 (not shown in the figure) aligns to the Beta60 amino acid codon and has the sequence 5'AAGGCAGTTATCACTTTCTTGCCATGGGCCTTCA. METHODS In separate reactions, DNA templates from strains of Hbbd haplotype (C3H/HeH, BALB/cJ, DBA/2J, 101/H), Hbbs haplotype (C57BL/6J, Mus spretus) and 20 of the 26 recombinant inbred (RI) strains of the BXD panel (C57BL/6J x DBA/2J) were prepared and tested for their ability to support amplification with each of the allele specific primers [1a]/[1b] as described (Jones & Peters, 1991). Spleen and lung from mice of the BXD panel were obtained as a gift from Dr I Lush. PCR was performed on lug genomic DNA in a final volume of 50ul containing 20 pmol of each primer. After heating to 95 degrees C for 10 mins 30 cycles of DNA synthesis were performed (1 min 94 degrees C, 1 min 55 degrees C and 2 min 72 degrees C, extended by 2 sec per cycle and extended to 10 minutes on the last cycle). Following amplification 20% of the reaction mix was subjected to electrophoresis in a 1.5% agarose gel containing 0.5ug/ml ethidium bromide. Fig 1 shows the sequences of the allele specific primers; [1a] specifies the Hbb-b2s allele and [1b] the Hbb-b2d allele. RESULTS & DISCUSSION Allele specific Primers. Detailed analysis of Hbb-bl and Hbb-b2 showed that there are very few sequence differences that could be used to type the alleles of one gene in the presence of the other closely related gene (eg to discriminate Hbb-b2s from Hbb-b2d in the presence of Hbb-b1). One appropriate sequence difference has been identified in the 5' untranslated region of Hbb-b2, at nucleotide number 15 from the cap site (Fig 1) At this position the nucleotide sequence is T in Hbb-b2s, G in Hbb-b2d and C in both Hbb-bl alleles (Fig 1). Primers [1a] and [lb], specific for Hbb-b1s alleles, have 3' termini that align to nucleotide 15 and differ from each other only in their 3' terminal nucleotide sequences (Fig 1); primer [1a] is complementary to the Hbb-b2s allele and primer [lb] to the Hbb-b2d allele. Both primers have a 3' terminal nucleotide that will produce a mismatch with Hbb-bl and have a deliberate mismatch (C/C) introduced close to the 3' terminus to ensure specificity (Newton et al., 1989; Kwok et a1 1990; Jones & Peters, 1991). The terminal nucleotide (T) of the Hbb-b2s specific primer [1a] will produce a T/C mismatch with Hbb-b2d and a T/G mismatch with Hbb-bl. Conversely, the terminal nucleotide (G) of primer [lb] will produce a G/A mismatch with Hbb-b2s and a G/G mismatch with Hbb-bl (Fig 1). Allele specific amplification of Hbbd and Hbbs DNA templates. The ability of each allele specific primer to support amplification was tested using duplicate samples of DNA from inbred strains of Hbbd (C3H/HeH , BALB/cJ, DBA/2J, and 101/H) and Hbbs (C57BL/6J, Mus spretus) haplotype. Successful amplification with either allele specific amplification primer together with primer [2] resulted in the production of a 398 bp fragment (Fig 2). As seen in Fig 2 amplification was allele specific; the Hbb-b2s primer [1a] supported amplification on Hbbs templates (Fig 2a tracks 2, 5) but when the DNA template was replaced with that from Hbbd haplotype (Fig 2a tracks 1, 3, 4, 6) the 398 bp amplification product was no longer observed. However, on substitution with primer [lb] amplification was seen on Hbbd DNA templates (Fig 2a tracks 1, 3, 4, 6) but not on Hbbs templates. To confirm that the variation was at Hbb, DNAs from the BXD recombinant inbred strain (RI) panel were tested for their ability to support amplification with each of the allele specific primers, [la] Hbb-b2s and [1b] Hbb-b2d. As seen from Fig 2b, by performing the reaction with either primer [la] Hbb-b2s or [1b] Hbb-b2d the samples are either positive for [1a] and negative for [1b], or, positive for [1b] and negative for [la]. The results of typing Hbb by PCR in the 20 RI's tested are in total agreement with results obtained by protein analysis (Wood & Taylor). Furthermore, segregation of the Mus spretus, Hbb-b2s allele has also been used in typing an interspecific (Mus musculus x MUS spretus) backcross panel in which the F1 female hybrid (C3H/HeH x M. spretus) was backcrossed to C3H/HeH (results not shown). In the past electrophoretic separation of the haemoglobin tetramer in blood provided a quick and inexpensive method for Beta-globin haplotype analysis. However, this method is probably not routinely available in many laboratories and furthermore, cannot be employed to type an interspecific backcross panel that has already been converted into DNA. Allele specific amplification, using primers that differ in a single nucleotide, provides an alternative method for the sensitive and rapid typing of murine Beta-globin haplotypes without the need for Southern blotting or for electrophoresis of the protein. In addition, the data so far indicate that the primers distinguish the Hbbs and Hbbd haplotypes irrespective of strain. The sensitivity, reproducibility and ease of performing allele specific amplification coupled with the versatility of primers suggests numerous applications in the field of molecular genetics. This technique has potential application in the analysis of any locus in which an allelic difference is known to exist at the protein or nucleotide level. REFERENCES Erhart, M.A., Simons, K.S. and Weaver, S. (1985). Mol. Biol. Evol. 2:304. Gilmam, J.G. (1976). Biochem. J., 159:294. Jones, J. and Peters, J. (1991). Biochemical Genetics Konkel, D.A., Maizel, J.V. and Leder, P. (1979). Cell, 18:865. Kwok, S., Kellog, D.E., McKinney, N., Spasic, D., Gola, L., Levinson, C. and Sninsky, J.J. (1990). Nucl. Acid Res., 18:999. Lewis, S.E., Johnson, F.M., Skow, L.C., Popp, D., Barnett, L.B. and Popp, R.A. (1985). Proc. Natl. Acad. Sci. USA., 82:5828. Newton, C.R., Graham, A,, Heptinstall, L.E., Powell, S.J., Summers, C., Kalsheker, N., Popp, R.A. (1973). Biochim. Biophys. Acta,, 303:52. Smith J.C. and Markham, A.F. (1989). Nucl. Acid Res., 17:2503. Tiemeier, D.C.S., Tilghman, S.M., Polsky, F.I., Seidman, J.G, Leder, A., Edgell, M.H. (1978). Cell 14:403-411. Wood A.W. & Taylor B.A., (1979). J. Biol. Chem. 254:5647. Fig 2 - (Legend). Agarose gel showing amplification products following amplification with the Hbb-b2 specific primers; [la] Hbb-b2s and [lb] Hbb-b2d. The primers used are indicated above the corresponding gels. DNA samples run on the gel shown on top have been amplified with primer [la] and those run on the gel shown below with primer [lb], Successful amplification produced a 398 bp band, which is indicated by an arrow. The marker track (m) contains a 1 kb ladder (Gibco-BRL). (2a) amplification of Hbbd and Hbbs DNA templates. The Hbbd and Hbbs haplotypes are indicated by d and s respectively, each track represents a DNA sample from (1) C3H/HeH; (2) C57BL/6J; (3) BALB/cJ; (4) DBA/2J; (5) Mus spretus and (6) 101/H. (2b) amplification of DNA templates from BXD RI lines. Track B is from C57BL/6J and D from DBA/2J; the RI lines are indicated by their number. The Hbb-b2 typing (d or s) for each RI line is shown beneath the gel. |
