Restriction Mapping

Abstract

Deoxyribonucleic acid is partially or completely digested with one or more restriction endonucleases. The generated fragments are separated by size and then ordered by combinatorial analysis to generate a physical map. The size of the fragments generated by a restriction endonuclease digest depends on the frequency of the enzyme's recognition sequence in the analysed genome. Resolution of the restriction map can thus be influenced by choosing the appropriate restriction enzyme and needs to be adapted to each species. The method has been used in the past to generate physical maps of plasmids, phages or whole genomes and is meanwhile employed to generate de novo genome assemblies by next‐generation sequencing of nonmodel organisms with polyploid genomes.

Key Concepts

  • Restriction enzymes generate DNA fragments at sequence‐defined genomic sites.
  • The average size of restriction digest‐generated DNA fragments can be controlled by choosing a restriction enzyme with rare (large fragments) or frequent (small fragments) recognition sequences.
  • Restriction fragment‐digested DNA can be used for next‐generation sequencing, which facilitates genomic mapping of nonmodel organisms with complex genomes.

Keywords: agarose gel electrophoresis; physical map; pulsed‐field gel electrophoresis; restriction endonuclease; Southern blot hybridisation

Figure 1. Detection of the large deletion CFTRdele2,3(21 kb) within the CFTR gene. CFTRdele2,3(21 kb) is a mutation that causes the monogenic disease cystic fibrosis. The above Southern blot was generated by pulsed‐field gel electrophoresis (PFGE) analysis: Agarose‐embedded DNA (deoxyribonucleic acid) samples prepared from fresh blood samples (see also: Genomic DNA: Purification) were partially digested with SalI (New England BioLabs) and separated by electrophoresis in a CHEF DR11 apparatus with 0.5 × TBE (0.045 M TRIS‐borate, 0.001 M EDTA, pH 8.3) at 10 °C, 6 V cm−1, and a 120° reorientation angle, with the following pulse times: two linear ramps from 5 to 30 s over 18 h and from 5 to 80 s over 20 h. The gel was stained with ethidium bromide, denatured, and transferred to Hybond N+ membranes (Amersham). CFTR fragments were detected by hybridisation with a radiolabelled CFTR cDNA (complementary deoxyribonucleic acid) probe spanning exons 7–24 (T16–4.5, ATCC). The sample of the heterozygous patient (lane 2) exhibits mobility shifts in two SalI fragments, which both are consistent with the presence of a deletion of 20–25 kb. Please note that the genomic sequence of the CFTR gene encompasses more than 200.000 bp. A large genomic deletion within the CFTR gene, inherited in the heterozygous state, can best be detected using a restriction enzyme with a rare recognition sequence that generates few (ideally one or two) fragments that can be stained with a genomic probe by Southern blot. Under these conditions, the wild‐type and the deleted gene variant will be represented side by side by one larger (wild‐type) and one smaller (deleted) signal. Reproduced with permission from Dörk et al. 2000 © Springer.
Figure 2. Combinatorial restriction digest using three restriction enzymes to generate a physical map of a P. aeruginosa clone C chromosome. After digestion of the agarose‐embedded DNA with PacI and SwaI, the fragments were separated by the contour‐clamped homogeneous electric field method; the running conditions were 3 V cm−1 with 1.0% agarose, and pulse times were increased in two linear ramps of from 52 to 67 s for 30 h and 60 to 380 s for 40 h. The whole lane was cut out, digested with SpeI, and separated in the second dimension; the running condition was 6 V cm−1, and pulse times were linearly increased in three ramps of from 8 to 50 s for 22 h, 12 to 25 s for 23 h and 1 to 15 s for 15 h. Complete single and double digests were applied as size markers to the outermost lanes to allow an assignment of restriction fragments: Lane 1 – SpeI‐SwaI‐PacI; Lane 2 – SpeI; Lane 3 – SpeI‐SwaI‐PacI; Lane 4 – SpeI‐SwaI‐PacI; Lane 5 – SpeI; Lane 6 – SpeI‐SwaI‐PacI; Lane 7 – SwaI‐PacI; Size marker: lambda DNA concatemers. As a consequence, PacI‐SwaI‐generated restriction fragments from the first dimension will be cut by SpeI into two, three or more smaller fragments if they contain one, two or more SpeI sites. If a PacI‐SwaI‐generated restriction fragment from the first dimension contains two SpeI restriction sites, one of the three resulting SpeI fragments can directly be identified from the SpeI‐only‐digest lane 2. Reproduced with permission from Schmidt et al. 1996 © American Society for Microbiology.
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References

Burns M, Starrett J, Derkarabetian S, et al. (2016) Comparative performance of double‐digest RAD sequencing across divergent arachnid lineages. Molecular Ecology Resources 17: 418–430.

Dörk T, Macek M Jr, Mekus F, et al. (2000) Characterization of a novel 21‐kb deletion, CFTRdele2,3(21 kb), in the CFTR gene: a cystic fibrosis mutation of Slavic origin common in Central and East Europe. Human Genetics 106 (3): 259–268.

Liu Z, Zhu H, Liu Y, Kuang J, et al. (2016) Construction of a high‐density, high‐quality genetic map of cultivated lotus (Nelumbo nucifera) using next‐generation sequencing. BMC Genomics 17: 466.

Schmidt KD, Tümmler B and Römling U (1996) Comparative genome mapping of Pseudomonas aeruginosa PAO with P. aeruginosa C, which belongs to a major clone in cystic fibrosis patients and aquatic habitats. J Bacteriol 178 (1): 85–93.

Shirasawa K, Tanaka M, Takahata Y, et al. (2017) A high‐density SNP genetic map consisting of a complete set of homologous groups in autohexaploid sweetpotato (Ipomoea batatas). Scientific Reports 7: 44207.

Further Reading

Bautsch W, Römling U, Schmidt KD, et al. (1997) Long‐range restriction mapping of genomic DNA. In: Dear PH (ed.) Genome Mapping – A Practical Approach, pp. 281–313. Oxford, UK: IRL/Oxford University Press.

Boseley PG, Moss T and Birnstiel ML (1980) 5′ Labelling and poly(dA) tailing. Methods in Enzymology 65: 478–494.

Danna AJ (1980) Determination of fragment order through partial digests and multiple enzyme digests. Methods in Enzymology 65: 449–467.

Römling U, Fislage R and Tümmler B (1996) Macrorestriction mapping and analysis of bacterial genomes. In: Birren B and Lai E (eds) Nonmammalian Genome Analysis, pp. 165–195. San Diego, CA: Academic Press.

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How to Cite close
Tümmler, Burkhard, and Stanke, Frauke(Jan 2018) Restriction Mapping. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0005363.pub2]