PCR-based genome walking methods (review)

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Abstract

The review discusses a range of classical and modern methods used to determine the nucleotide sequence of unknown DNA regions flanking known ones. These methods are applied to decipher the regulatory regions of genes, identify integration sites of T-DNA or viruses, and so on, in cases where the use of whole-genome sequencing is not justified. To amplify a DNA segment, a binding site for a primer must be added to the end of the unknown sequence. This can be achieved either by ligating an adapter or by annealing a degenerate primer under gentle conditions, or by looping the DNA fragment so that the target region is surrounded by known sequences. The second important task is to eliminate the inevitable products of nonspecific binding of adapters or degenerate primers, which is often resolved through multiple rounds of nested PCR. Different methods vary significantly in terms of complexity, prevalence, and the availability of required reagents.

About the authors

Elena S. Okulova

Saint Petersburg State University; All-Russian Institute of Plant Protection

Author for correspondence.
Email: elenaok.advert@gmail.com
ORCID iD: 0009-0001-7349-8925
SPIN-code: 7166-0090

Master of Science, Research Associate, Department of Genetics and Biotechnology

Russian Federation, 7/9 Universitetskaya emb., Saint Petersburg, 199034; Saint Petersburg

Mikhail S. Burlakovskiy

Saint Petersburg State University

Email: burmish@yandex.ru
ORCID iD: 0000-0001-6694-0423
SPIN-code: 3679-0860

PhD, Junior Researcher, Department of Genetics and Biotechnology

Russian Federation, 7/9 Universitetskaya emb., Saint Petersburg, 199034

Ludmila A. Lutova

Saint Petersburg State University

Email: la.lutova@gmail.com
ORCID iD: 0000-0001-6125-0757
SPIN-code: 3685-7136
Scopus Author ID: 6603722721

Dr. Sci. (Biol.), professor

Russian Federation, 7/9 Universitetskaya emb., Saint Petersburg, 199034

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Supplementary files

Supplementary Files
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1. JATS XML
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2. Figure 1. Schematic representation of the EPTS/LM-PCR principle (based on Schmidt et al., 2001)
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3. Figure 2. Schematic representation of the Panhandle-PCR principle (based on Douglas et al., 1992)
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4. Figure 3. Schematic representation of the boomerang-PCR principle (based on Hengen, 1995)
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5. Figure 4. Schematic representation of the principle of adapter ligation PCR (based on O'Malley et al., 2007)
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6. Figure 5. Schematic representation of the principle of restriction site extension PCR (based on Ji et al., 2010)
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7. Figure 6. Schematic representation of the principle of Template-blocking PCR (based on Bae and Sohn, 2010)
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8. Figure 7. Schematic representation of the principle of SSP-PCR (based on Shyamala and Ames, 1989)
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9. Figure 8. Schematic representation of the principle of restriction site PCR (based on Sarkar, Turner and Bolander, 1993)
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10. Figure 9. Schematic representation of the principle of the FPNI-PCR method (based on Wang et al., 2011)
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11. Figure 10. Schematic representation of the principle of the SWPOP-PCR method (based on Chang et al., 2018).
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12. Figure 11. Schematic representation of the principle of the PST-PCR method (based on Kalendar, Shustov and Schulman, 2021)
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13. Figure 12. Schematic representation of the principle of the SLRA-PCR method (based on Li, Fu and Li, 2019
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14. Figure 13. Schematic representation of the principle of the FPR-PCR method (based on Pei et al., 2022)
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15. Figure 14. Schematic representation of the principle of the wristwalch PCR method (based on Wang et al., 2022)
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16. Figure 15. Schematic representation of the principle of a restriction-independent method for cloning genomic DNA segments beyond known sequences (based on Rudi, Fossheim and Jakobsen, 1999)
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17. Fig. 1. Schematic representation of the principle of inverted PCR (based on E.K. Hui et al. [6]). Solid line — known DNA sequence; dashed line — unknown DNA region; arrow — primer binding site; white rectangles — restriction sites. DNA is cleaved with a restriction enzyme that does not have a cutting site in the integrating region, looped under conditions favorable for the formation of monomeric rings, and amplified. PCR uses primers in different directions that are complementary to the ends of the integrating fragment.

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18. Fig. 2. Schematic representation of the principle of ligation-mediated PCR. Solid line — known DNA sequence; dashed line — unknown DNA region; dotted line — amplification product; small arrow — primer binding site; black rectangles — adapter

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19. Fig. 3. Schematic representation of the vectorette PCR principle (based on E.K. Hui et al. [6]). Solid line — known DNA sequence; dashed line — unknown DNA region; shaded arrow — primer binding site to the vectorette; shaded region — DNA fragment complementary to the primer to the vectorette; black arrow — primer binding site to the target DNA. DNA is cleaved with a restriction enzyme to form a sticky 5' end. Then, a synthetic oligonucleotide (linker), called a vectorette, is ligated to the 5' end. PCR amplification of the DNA fragment is carried out using an internal primer specific for the target DNA and a primer specific for the vectorette.

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20. Fig. 4. Schematic representation of the structures of the vectorette and splinkerett cassettes (based on E.K. Hui et al. [6])

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21. Fig. 5. Schematic representation of the principle of capture PCR (CPCR) (based on M. Lagerstrom et al. [10]). Solid line — genomic DNA; black rectangles — adapter; arrow — primer entry site; B — biotin. The first strand is synthesized based on a single gene-specific biotinylated primer, which allows fixation of this fragment on a streptavidin-coated substrate. Unlabeled DNA is removed during washing. The target fragment is amplified with a primer to the adapter and a second specific primer

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22. Fig. 6. Schematic representation of the T-linker PCR principle (based on Y. Yuanxin et al. [15]). Solid line — known DNA sequence; dashed line — unknown DNA region; arrow — primer binding site; black rectangles — linker; S1, S2, and S3 — specific primers binding to the known sequence of the target molecule; W1 and W2 — walking primers binding to the T-linker sequence; A — A-“tail” of the target molecule; T — T-nucleotide of the T-linker; Δ — expected difference in the amplification products with specific primers S2 and S3 in separated second-cycle reactions

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23. Fig. 7. General scheme of primer arrangement in PCR with random primers. Solid line — known DNA sequence; dashed line — unknown DNA region; arrows — primer landing sites

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24. Fig. 8. Schematic representation of the principle of the UFW method (based on K.W. Myrick and W.M. Gelbart [21]). Solid line – known DNA sequence; dashed line – unknown DNA region; dotted line – amplification product; short arrows with numbers – UFW primers, numbered in order of use.

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25. Fig. 9. Schematic representation of the principle of the SiteFinding PCR method (based on G. Tan et al. [22]). Solid line – known DNA sequence; dashed line – unknown DNA region; arrow – primer binding site; white rectangle – restriction site; GSP – gene-specific primers, SFP – SiteFinding primers

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26. Fig. 10. Schematic representation of the principle of the TAIL-PCR method (based on Y.-G. Liu et al. [23]). Solid line — known DNA sequence; dashed line — unknown DNA region; arrow — primer binding site; TR1, TR2, TR3 — nested primers complementary to the known sequence; AD — short arbitrary degenerate primers (15–16 bp) with low melting temperature and varying degrees of degeneracy. By alternating the annealing temperature from high (62 to 68 °C) in high-fidelity cycles to low (44 °C) in low-fidelity cycles, the relative efficiency of amplification of specific and non-specific products is thermally controlled

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27. Fig. 11. Schematic representation of the principle of the POP-PCR method (based on H. Li et al. [26]). Solid line – known DNA sequence; dashed line – unknown DNA region; arrow – primer landing site.

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28. Fig. 12. Schematic representation of the RCA–GIP principle (based on A. Tsaftaris et al. [36]). Black line – genomic DNA; arrows – random hexamer primers; dotted lines – concatemer copies.

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29. Fig. 13. Schematic representation of the 4SEE principle. The shaded circle is the region of DNA cross-linking with formaldehyde; the gray line is the sequence of known DNA; the black line is the unknown region of DNA; the white and gray rectangles are restriction sites.

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