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

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2. Fig. 1. Schematic representation of inverted PCR (based on E.K. Hui et al. [6]). Solid line, known DNA sequence; dashed line, unknown DNA segment; arrow, primer binding site; white rectangles, restriction sites. DNA is cleaved by a restriction enzyme that does not have a cutting site within the insert, then circularized under conditions favorable for the formation of monomeric circles and amplified. In PCR, primers complementary to the ends of the insert fragment are used in opposite directions

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3. Fig. 2. Schematic representation of PCR mediated by ligation. Solid line, known DNA sequence; dashed line, unknown DNA segment; dotted line, amplification product; small arrow, primer binding site; black rectangles, adapters

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4. Fig. 3. Schematic representation of the “vectorette PCR” (based on E.K. Hui et al. [6]). Solid line, known DNA sequence; dashed line, unknown DNA segment; hatched arrow, primer binding site to the “vectorette”; hatched segment, DNA fragment complementary to the “vectorette” primer; black arrow, primer binding site to the target DNA. DNA is cleaved by a restriction enzyme, generating a 5'-sticky end. Then, a synthetic oligonucleotide (linker) called “vectorette” is ligated to the 5'-end. PCR amplification of the DNA fragment is performed using an internal primer specific to the target DNA and a primer specific to the “vectorette”

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5. Fig. 4. Schematic representation of the structures of «vectorette» and «splinkerette» cassettes (based on E.K. Hui et al. [6])

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

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7. Fig. 6. Schematic representation of the T-linker PCR (based on Y. Yuanxin et al. [15]). Solid line, known DNA sequence; dashed line, unknown DNA segment; 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, presumed difference in amplification products with specific primers S2 and S3 in separate reactions of the second cycle

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8. Fig. 7. The general scheme of primer placement in PCR with random primers. Solid line, known DNA sequence; dashed line, unknown DNA segment; arrows, primer binding sites

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

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

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11. Fig. 10. Schematic representation of the TAIL-PCR method (based on Y.-G. Liu et al. [23]). Solid line, known DNA sequence; dashed line, unknown DNA segment; 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. Alternating annealing temperatures from high (62 to 68 °C) in high stringency cycles to low (44 °C) in low stringency cycles thermally controls the relative efficiency of amplification of specific and nonspecific products

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12. Fig. 11. Schematic representation of the POP-PCR (based on H. Li et al. [26]). Solid line, known DNA sequence; dashed line, unknown DNA segment; arrow, primer annealing site

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13. Fig. 12. Schematic representation of the RCA-GIP method (based on A. Tsaftaris et al. [36]). Black line, genomic DNA; arrows, random hexameric primers; dotted lines, copies of concatemers

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14. Fig. 13. Schematic representation of the 4SEE principle. Shaded circle, DNA crosslinking region with formaldehyde; gray line, known DNA sequence; black line, unknown DNA segment; white and gray rectangles: restriction sites

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15. Изображение 1. Схематическое изображение принципа EPTS/LM-PCR (на основе Schmidt et al., 2001)
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16. Изображение 2. Схематическое изображение принципа «Panhandle»-PCR (на основе Douglas et al., 1992)
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17. Изображение 3. Схематическое изображение принципа «boomerang»-PCR (на основе Hengen, 1995)
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18. Изображение 4. Схематическое изображение принципа ПЦР с лигированием адаптера (на основе O'Malley et al., 2007)
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19. Изображение 5. Схематическое изображение принципа ПЦР с удлинением сайта рестрикции (на основе Ji et al., 2010)
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20. Изображение 6. Схематическое изображение принципа Template-blocking PCR (на основе Bae and Sohn, 2010)
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21. Изображение 7. Схематическое изображение принципа SSP-PCR (на основе Shyamala and Ames, 1989)
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22. Изображение 8. Схематическое изображение принципа ПЦР сайта рестрикции (на основе Sarkar, Turner and Bolander, 1993)
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23. Изображение 9. Схематическое изображение принципа метода FPNI-PCR (на основе Wang et al., 2011)
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24. Изображение 10. Схематическое изображение принципа метода SWPOP-PCR (на основе Chang et al., 2018).
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25. Изображение 11. Схематическое изображение принципа метода PST-PCR (на основе Kalendar, Shustov and Schulman, 2021)
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26. Изображение 12. Схематическое изображение принципа метода SLRA-PCR (на основе Li, Fu and Li, 2019)
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27. Изображение 13. Схематическое изображение принципа метода FPR-PCR (на основе Pei et al., 2022)
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28. Изображение 14. Схематическое изображение принципа метода wristwalch PCR (на основе Wang et al., 2022)
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29. Изображение 15. Схематическое изображение принципа независимого от рестрикции метода клонирования сегментов геномной ДНК за пределами известных последовательностей (на основе Rudi, Fossheim and Jakobsen, 1999)
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