Rapid determination of sequences flanking microsatellites

Martin Yuille

Rapid determination of sequences flanking microsatellites

Nucleic Acids Research, 1991

1950 Nucleic Acids Research, Vol. 19, No. 8 Rapid determination of sequences flanking microsatellites M.A.R.Yuille, D.R.Goudie, N.A.Affara and M.A.Ferguson-Smith University of Cambridge, Department of Pathology, Tennis Court Road, Cambridge CB2 1QP, UK Submitted February 15, 1991 Microsatellites are repeated simple sequences found in genomic DNA that may, for example, consist of alternate cytosine and adenine bases. If the most 3' base of such a microsatellite is dA, then its sequence may be written as (5'dC-dA3')n.(5'dT-dG3')n. A method is described here that uses a set of oligonucleotides of which one will prime a sequencing reaction efficiently to yield the sequence of the immediate flank of a cloned microsatellite previously identified by Southern blot hybridisation. An oligonucleotide composed of the repeat motif and the appropriate non-motif 3' base should prime a sequencing reaction proceeding into one flank of a given microsatellite. There are six combinations of repeat motif and non-motif 3' base. Three are of the form (5'dG-dT3')mdX (where X = A, C or T) and three of the form (5'dT-dG3')ndY (where Y = A, C or G). A corresponding set of six primers could be used to sequence into the other flank. These would be of the form (5'dC-dA3')mdX' (where X' = A, G or T) and (5'dA-dC3')mdY' (where Y' = C, G or T). Sequencing was performed using the Pharmacia T7 polymerase kit and followed manufacturer's instructions but best results were obtained when all steps were performed at 37°C. Two plasmid templates known to contain cytosine/adenine microsatellites (by probing a Southern blot with end-labelled (5'dG-dT3')15 and washing to 0.5 x SSC at 55°C) were tested: pASSG1 (whose sequence had been previously obtained conventionally (Yuille et al., 1990) indicating that (5'dG-dT3')7A would give unambiguous sequencing data into one flank) and pT512 (a 1.7 kb insert plasmid subclone of a cosmid obtained by screening a library with a fragment of pTHH22 (Holm et al., 1988)). For each plasmid, six sequencing reactions were performed using primers of the form (5'dG-dT3')7dX and (5'dTdG3')7dY. As predicted, for each plasmid, only one primer gave unambiguous sequencing data (Fig. 1). In the case of pT512, a primer was then designed from the sequence obtained and used to sequence back toward and through the microsatellite. Ambiguous data may arise if more than one microsatellite of the same type is present in one clone. However, in the pT512 sequence we have obtained, there are two (5 'dA- dC3')2dC.dG(5'dG-dT3')2 microsatellites and these fail to obscure the signal. Theoretically, 35 out of 36 clones containing two large microsatellites should give unambiguous data from one flank of each microsatellite if all 12 primers are employed. This method should allow determination of the flanking sequences of any simple repetitive sequence in one (or two) experiments. ACKNOWLEDGEMENTS We thank Mr J. Lyall for technical assistance and Dr C. A. Sargeant for access to a cosmid library. This work was supported by the MRC Human Genome Mapping Project and the Imperial Cancer Research Fund. REFERENCES 1. Holm,T., et al. (1988) Nucl. Acids Res. 15, 5216. 2. Yuille,M.A.R., et al. (1990) Nuci. Acids Res. 18, 2472. ! "I .4 .>~f I) 9 ( i- {I " _ ; 4 ] n h 7 8 t) {1 w2,> ,s. .. id ._ _ _ :i ; Y':: : .5E.. -- _ .. ._'Mf:S ''Y<F<2xO*.*: s .. -_:. - _ ..S,!. ..... j_ ,. r: s-! - - - K ;.::*-...-* . *1) 8S -) 222 I2 3 4 Figure 1. Sequencing reactions from the 3' end of the cytosine/adenosine strand of microsatellites in pT512 (A) and in pASSG1 (B). In A, primers (5'dG- dT3')7dC,(5'dG-dT3')7T, (5'dT-dG3')7A, (5'dT-dG3')7C and (5'dT-dG3')7G were used in lanes 1-4, 5-8, 9-12, 13-16, 17-20 respectively ((5'dG- dT3')7dA not shown). In B, primers (5'dT-dG3')7G, (5'dG-dT3')7A, (5'dG- dT3')7C, (5'dG-dT3')7T, (5'dT-dG3')7A and (5'dT-dG3')7C were used in lanes 1-4, 5-8, 9-12, 13-16, 17-20, 21-24 respectively. Termination reactions A, C, G and T were loaded consecutively in each set of four lanes. l. 1991 Oxford University Press

.j_ l. 1991 Oxford University Press 1950 Nucleic Acids Research, Vol. 19, No. 8 Rapid determination of sequences flanking microsatellites M.A.R.Yuille, D.R.Goudie, N.A.Affara and M.A.Ferguson-Smith University of Cambridge, Department of Pathology, Tennis Court Road, Cambridge CB2 1QP, UK Submitted February 15, 1991 Microsatellites are repeated simple sequences found in genomic DNA that may, for example, consist of alternate cytosine and adenine bases. If the most 3' base of such a microsatellite is dA, then its sequence may be written as (5'dC-dA3')n.(5'dT-dG3')n. A method is described here that uses a set of oligonucleotides of which one will prime a sequencing reaction efficiently to yield the sequence of the immediate flank of a cloned microsatellite previously identified by Southern blot hybridisation. An oligonucleotide composed of the repeat motif and the appropriate non-motif 3' base should prime a sequencing reaction proceeding into one flank of a given microsatellite. There are six combinations of repeat motif and non-motif 3' base. Three are of the form (5'dG-dT3')mdX (where X = A, C or T) and three of the form (5'dT-dG3')ndY (where Y = A, C or G). A corresponding set of six primers could be used to sequence into the other flank. These would be of the form (5'dC-dA3')mdX' (where X' = A, G or T) and (5'dA-dC3')mdY' (where Y' = C, G or T). Sequencing was performed using the Pharmacia T7 polymerase kit and followed manufacturer's instructions but best results were obtained when all steps were performed at 37°C. Two plasmid templates known to contain cytosine/adenine microsatellites (by probing a Southern blot with end-labelled (5'dG-dT3')15 and washing to 0.5 x SSC at 55°C) were tested: pASSG1 (whose sequence had been previously obtained conventionally (Yuille et al., 1990) indicating that (5'dG-dT3')7A would give unambiguous sequencing data into one flank) and pT512 (a 1.7 kb insert plasmid subclone of a cosmid obtained by screening a library with a fragment of pTHH22 (Holm et al., 1988)). For each plasmid, six sequencing reactions were performed using primers of the form (5'dG-dT3')7dX and (5'dTdG3')7dY. As predicted, for each plasmid, only one primer gave unambiguous sequencing data (Fig. 1). In the case of pT512, a primer was then designed from the sequence obtained and used to sequence back toward and through the microsatellite. Ambiguous data may arise if more than one microsatellite of the same type is present in one clone. However, in the pT512 sequence we have obtained, there are two (5 'dAdC3')2dC.dG(5'dG-dT3')2 microsatellites and these fail to obscure the signal. Theoretically, 35 out of 36 clones containing two large microsatellites should give unambiguous data from one flank of each microsatellite if all 12 primers are employed. This method should allow determination of the flanking sequences of any simple repetitive sequence in one (or two) experiments. ACKNOWLEDGEMENTS We thank Mr J. Lyall for technical assistance and Dr C. A. Sargeant for access to a cosmid library. This work was supported by the MRC Human Genome Mapping Project and the Imperial Cancer Research Fund. REFERENCES 1. Holm,T., et al. (1988) Nucl. Acids Res. 15, 5216. 2. Yuille,M.A.R., et al. (1990) Nuci. Acids Res. 18, 2472. ! "I .4 .>~f I) 9i- ({I _" ; 4 ] n h 7 8 t) {1 w2,> ,s. .. ._ id _ _ :i ; Y':: : .5E.. -- _ .. ._'Mf:S ''Y<F<2xO*.*: s .. -_:. - _ ..S,!. ,. r: - s-! - - K ;.::*-...-* . 222-) I2 3 4 *1) 8S Figure 1. Sequencing reactions from the 3' end of the cytosine/adenosine strand of microsatellites in pT512 (A) and in pASSG1 (B). In A, primers (5'dGdT3')7dC,(5'dG-dT3')7T, (5'dT-dG3')7A, (5'dT-dG3')7C and (5'dT-dG3')7G were used in lanes 1-4, 5-8, 9-12, 13-16, 17-20 respectively ((5'dGdT3')7dA not shown). In B, primers (5'dT-dG3')7G, (5'dG-dT3')7A, (5'dGdT3')7C, (5'dG-dT3')7T, (5'dT-dG3')7A and (5'dT-dG3')7C were used in lanes 1-4, 5-8, 9-12, 13-16, 17-20, 21-24 respectively. Termination reactions A, C, G and T were loaded consecutively in each set of four lanes.

The Malayalam film Kumbalangi Nights (2019) received wide appreciation as a mainstream/popular movie which decoded masculinity. This article attempts a close reading of the film to see how the film unsettles the usual representation of 'the hero' in Malayalam/Indian films. It argues that Kumbalangi Nights can actually be read as a satire of the filmic hero usually seen in the earlier decades' Superstar films in Malayalam. It argues that what Kumbalangi Nights does is to show that the 'hegemonic masculinity' which hero centric popular Malayalam/Indian films celebrate is actually 'toxic masculinity'. The institution of family is quite central in keeping the gender divisions intact. The article also tries to point out that our set understanding about the 'proper' middle class family as the space of ideal domestic contentment, as usually depicted in popular movies, is shattered in Kumbalangi Nights. It also tries to delineate how the agency of women is central to the narrative of the film in the unravelling of toxic masculinity and in problematising power relations within the family. The article argues that the film shows the inside of the ideal middle class household as a place of power play, where women are most often living in constant fear; it is the space of the most violent attacks-physical and emotional-against women and children. It reads that Kumbalangi Nights offers an alternate model of family based on love, empathy, kindness and virtue rather than relation through body fluids. The article is divided into five sections: the first section gives the theoretical framework of the article, the second section introduces the plot and setting of the movie, the third section-Men of Kumbalangi and Masculinity-is further divided into two parts which look at the film's undoing of the concept of hero and hegemonic masculinity and its portrayal of other masculinities. The fourth section presents a critique of the normative middle class family in the film. The fifth section analyses the characterization of women in the film and its importance in the undoing of the hegemonic masculinity and power play in the normative family. The final section concludes the article by briefly placing Kumbalangi Nights against the Malayalam Superstar films of the 1990s and 2000s to bring out the contrast and ends by opening up further possibilities of research on the film.

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Rapid determination of sequences flanking microsatellites