Identification of unknown nucleotide sequences flanking already characterized DNA regions can be pursued by number of different PCR- based methods commonly known as Genome walking (GW) GW methods have been developed in the last 20 years, with continuous improvements added to the first basic strategies First reported by Hengen in 1995 in comparison with other technologies Hui et al., in 1998 reviewed in detail The extreme flexibility of GW strategies makes its application possible in every standardly equipped research laboratory. In addition, the possibility of merging GW strategies to next generation sequencing approaches will undoubtedly extend the future application of this by now basic technique of molecular biology.

2. Source: National plant Genome Research Institute.

3. UNIVERSITY OF HORTICULTURAL SCIENCES,
BAGALKOT
Prakash G
UHS14PGM416

4. 4

5. 5

6. Identification of unknown nucleotide sequences flanking already
characterized DNA regions can be pursued by number of different
PCR- based methodscommonly known asGW
GW methods have been developed in the last 20 years, with
continuousimprovementsadded to thefirst basic strategies
First reported by Hengen in 1995 in comparison with other
technologies
Hui etal., in 1998 reviewed in detail
6

7. In terms of high throughput DNA sequencing technology, when
more than 1000 genomes have been completely sequenced.
GW methods and improvements of several available strategies
continue to be published with a steady positive trend.
So, for such constant interest can be found :
1. In the relatively low difficulty of different strategies, which do not
require expensive equipment or highly trained personnel.
2. Increasing possibilities of applying GW methods to eukaryotic
genomes.
7

8. Supporting points
Inverse PCR:
• Used to amplify the sequences flanking a segment the border
sequences of which are known.
• The target DNA is cut with a restriction enzyme that produces
sticky ends does not cut within region of known sequence.
8

9. Nested PCR:
The target sequence is amplified using a pair of PCR primers and
portion of the amplification product is re amplified using another
pair of PCR primers complementary to the regions located
immediately beyond the 3`- ends of the first pair of primers.
9

10. Restriction based (R-W)
Primer based (P-GW)
Extension based (E-GW)
10

11. • R-GW methods require a preliminary digestion of the
genomic DNA by suitable Restriction enzymes.
• Whose sites must be located at a proper distance from
the boundary between known and unknown sequences
• The Restriction fragments can be either self-
circularized or ligated.
11

12. Leoni et al. 2011
12

13. • P-GW, in which PCR amplifications are directly
carried out using a variously designed
combination(random or degenerate primer) coupled
to a sequence specific primer.
13

14. E-GW, in which the extension of a sequence
specific primer and subsequent 3` tailing of the
resulting single strand DNA provide the substrate
for the final PCR amplification.
14

15. Leoni et al. 2011 15

16. Protocol
To start, you need:
-the DNA sequence of a small region of the chromosome
-An adaptor: a small piece of DNA, 10-15 nucleotides long and double stranded of
which you also know the sequence. The adaptor can be ordered from a lab. This will
be ligated to fragments of the sequence after digestion.
-To order PCR primers against the known region of the gene and your small piece of
DNA
Known region of the
gene
Adaptor
Primers
16

17. Preparation of the DNA:
A few digestion reactions need to be set, each using a restriction enzyme
which will cut DNA at different place (i.e. different group of 6
nucleotide).
For simplicity, we will show only 2 in here.
Enzyme 1 cuts there:
Enzyme 2 cuts there:
17

18. Cont....
So we get two tubes, containing the same DNA, but this DNA has been cut
in different places:
Enzyme
1
Enzyme
2
18

19. Enzyme
1
Enzyme
2
LigaseLigase
Cont....
Then we add lots of adaptors and a ligase enzyme which is going to bind the
adaptor to the end bits of the DNA fragments
19

20. Enzyme
1
Enzyme
2
Cont....
20

21. Enzyme
1
PCR on the products of digestion by enzyme 1
Cont....
21

22. PCR on the products of digestion by enzyme 1
This fragment (100-1500bp)
can be easily sequenced
Cont....
Enzyme
1
22

23. Enzyme
1
Enzyme
2
This shows the DNA sequence that we know now:
This part of
the
new DNA
sequence can
be used to
design new
primers
Cont....
23

24. PCR on the products of digestion by enzyme 2
Enzyme
2
New primer, it could have only
been designed after getting
new DNA sequence from the
product of the first PCR
This fragment can be easily
sequenced, as a result, we
obtain a small fragment of
new sequence (100-1500bp)
Cont....
24

25. You can now go back to the products of the digestion by enzyme
1 and design new primers to walk further…
Extended know region of DNA sequence
against Which new primers can be
designed!!!!
Enzyme
1
Cont....
25

26. GW-Kits and customer services
Leoni et al. 201126

27. GW-patents
• The development of so many GW methods gave rise to the
application of numerous patents, claiming either a
methodological innovation of the process or the application
of a GW strategy for the solution of a specific problem.
• Patent retrieval was performed by using the Orbit
(http://www.orbit.com) platform (Questel, Paris), a web
resource specialized in intellectual property.
Leoni et al. 201127

28. Area of
interest
• Molecular
objectives
• Applications
De novo
sequencing
Insertional
mutagenes
is
Virus
integration
Transposons
T-DNA
Gene
identification
Nucleotide
Modification/mu
tation
Gene marking
Gene therapy
Retrovirus
studies
Transgenic
plants
Regulatory regions
Metagenomics
Multi gene
families
28

29. A large number of investigations employing GW strategies have
been developed to identify and characterize the insertion sites of
retroviral cDNAs or retrovirus derived vectors in the human
genome.
The first analysis by GW of a retroviral-mediated gene marking of
bone marrow cells used for autologous transplantion in patients
with neuroblastoma was reported by (Rill et al., 1994)
Viral integration
29

30. Gene marking proceded the use of retroviral derived vectors in
gene therapy as delivery vehicles of therapeutic genes. Most of
their genes in order to prevent dangerous infection.
Insertional mutagenesis can disrupt important genes, such as
those involved in the control of cell growth and division, leading
to cancer onset.
Additionally, introduced viral promoters and enhancer can
activate transcription of proto-oncogenes.
30

31. TRANSPOSO
NS
Transposon insertional mutagenesis is a basic tool for addressing gene
function through analysis of mutant phenotypes and identification of
mutated genes in eukaryotic genomes.
GW has contributed significantly to the analysis of transposition events,
providing valuable data for reverse genetic analysis and gene inactivation.
31

32. Transposon Plant GW approach ⁄ kit References
Ac ⁄ Ds Transgenic tobacco I-PCR Feschotte et al. (2002)
A. thaliana Long et al. (1993)
Tomato Meissner et al. (2000)
Maize Kolkman et al. (2005)
Lotus japonicus Tirichine et al. (2005)
dTph1 Petunia hybrida I-PCR Souer et al. (1995)
En A. thaliana I-PCR Aarts et al. (1993)
En ⁄ Spm-like Zingeria
biebersteiniana
DNA Walking
SpeedUp kit
Altinkut et al. (2006)
Antirrhinum majus Genome Walker kit Roccaro et al. (2007)
Mu Maize DLA GW Liu et al. (2009)
Ty-1 Apple Site finding PCR Zhao et al. (2007)
Transposon analysis in plant genomes by GW approaches.
Leoni et al. 201132

33. T-DNA
• Before field trials of genetically modified crops it is of primary
importance both to identify T-DNA insertion sites in the host
genome and to select transformed plants carrying a single T-DNA
copy (necessary for avoiding possible transgene silencing processes
activated by multiple T-DNA insertions).
• Spertini and co workers analysed the complexity of the T-DNA
integration pattern in transgenic Arabidopsis plants by analysing the
PCR pattern obtained by applying the GW method.
33

34. Objective: Agrobacterium rhizogenes and Agrobacterium tumefaciens are
plant pathogenic bacteria capable of transferring DNA fragments [transfer
DNA (T-DNA)] bearing functional genes into the host plant genome.
This naturally occurring mechanism has been adapted by plant
biotechnologists to develop genetically modified crops.
Department of Molecular Biotechnology, Ghent University, 9000 Ghent, Belgium;
International Potato Center, Lima 12, Peru, china.
34

35. Fig. 1. Organization of IbT-DNA1 and IbT-DNA2 in the genome of sweet potato. (A) IbT-
DNA1 of landrace “Huachano,” including ORFs showing significant homology to iaaM,
iaaH, C-prot, and Acs and a truncated iaaM in inverted orientation. This T-DNA is located in
an intron of a gene showing strong homology to plant F-box genes. The regions with
significant similarity to plant sequences is shown as a yellow line. (B) IbT-DNA2 of
“Huachano,” including ORFs with significant homology to ORF14, ORF17n, RolB/RolC,
ORF13, and ORF18/ORF17n. 35

36. Fig. 2. Southern blot analyses showing the integration of IbT-
DNA1 and IbTDNA2 into the sweet potato genome. Total
genomic DNA of landrace “Huachano” was digested with
Spe 1 and hybridized with various probes.
(A) Probe 1 complementary to the ORF coding for C-protein of
IbT-DNA1 revealing the presence of multiple (four estimated)
insertions into the sweet potato genome.
(B) Probe 2 complementary to the F-box gene in the region
flanking IbT-DNA1, revealing the presence of probably six copies,
four of which appear to correspond to similar bands in the
hybridization with probe 1 for IbTDNA1.
(C) Probe 3 complementary to the ORF coding for ORF17n of
IbT-DNA2.
Kyndt et al. 2015 36

37. Fig. 3. Phylogenetic trees generated by neighbor joining of (A) iaaM (399- nt fragment)
and (B) ORF13 (722-nt fragment) alignments. Values at the nodes indicate percentage of
bootstrap support (of 1,000 bootstrap replicates) and are indicated if greater than 50.
37

38. Fig. 4. Relative expression of four ORFs on IbT-DNA1 and two ORFs
located on IbT-DNA2. The figure shows the relative presence of mRNA in
different tissues of sweet potato, based on qRT-PCR.
38

39. GW approach ⁄ kit Plant References
Suppression PCR Arabidopsis Devic et al. (1997)
Potato Cottage et al. (2007)
Tobacco Cottage et al. (2007)
Shallot Zheng et al. (2001)
Grapefruit Rai (2006)
Subtractive PCR Banana Perez et al. (2006)
fingerprinting PCR Canola Taverniers et al. (2005)
I-PCR Tomato Knapp et al. (1994)
Maize Ronning et al. (2003)
T-linker PCR Rice Yuanxin et al. (2003)
TAIL-PCR Maize Yang et al. (2005)
APAgene GOLD Potato Cullen et al. (2011)
DNA Walking
SpeedUp
Potato Cullen et al. (2011)
Universal
vectorette
Potato Cullen et al. (2011)
Table 1.Analysis of T-DNA transgenes in plant genomes by GW
approaches.
39

40. Transgenic plants
40

41. Objective: The potential presence of unauthorised GMOs is assessed by the qPCR
SYBRGreen technology targeting the terminator 35S pCAMBIA element. its presence is
confirmed via the characterisation of the junction between the transgenic cassette and the
rice genome.
41

42. 42

43. Fig. 5. DNA walking strategy. (A) Designed primer position of t35S pCAMBIA a-R, t35S pCAMBIA
b-R and t35S pCAMBIA c-R to target t35S pCAMBIA sequence from t35S pCAMBIA 1300 sequence.
This sequence is identical for all pCAMBIA.
In the first step, single strand DNA (ssDNA) fragments are produced by a single primer extension
reaction using t35S pCAMBIA a-R primer. In the second step, four different DRT primers (A–D) are
immediately added individually to the four reaction tubes.
Fraiture et al. 2013
43

44. Fraiture et al. 2013 44

45. Characterisation of the junction between the integrated transgenic
pCAMBIA cassette and the rice genome.
Fig. 6. Visualisation of the amplicons obtained with
the different DRT mixes (A–D) on a 1% agarose gel.
Fraiture et al. 2013 45

46.  Fig. 7.
 Amplicon sequences presenting the junction between the pCAMBIA 1300 vector
(underlined) and the rice genome identified on the chromosome II and the chromosome
III, respectively.
 The t35S pCAMBIA c-R (in bold) and the UAP-N1/UAP-N2 primers are dotted-
underlined. These sequences were obtained by classic sequencing of the plasmids.46

47. Objective: Different methodologies for the determination of insertion sites using a
range of published protocols and commercially available kits were assessed in
transgenic lines of varying degrees of complexity, from low copy number to complex re-
transformed and co-transformed lines.
47

48. Table 2. Summary of transgenic potato lines selected for obtaining genomic
DNA flanking the transgene insertion site by various DNA walking methods.
Cullen et al. 2011
48

49. Table 3. Results summary of DNA walking methods for single
copy potato transgenic lines
Cullen et al. 2011 49

50. Fig.8. Amplification of T-DNA left border flanking regions from a single copy line
of transgenic potato (Bkt1-2) using the DNA Walking SpeedUp and GOLD Genome
Walking Kit.
Lane 1, 1 kb DNA ladder, 250–10,000 bp; Final round nested PCR products: lanes
2-5 ACP1 to ACP4 SpeedUpTM primers, respectively; lanes 6–9 DRTA to DRTD
APAgeneTM primers, respectively. * All bands excised and purified using WizardÒ
Gel Clean-Up System (Promega) for direct sequencing
Cullen et al. 2011
50

51. 9
51

52. Table 4. Results summary for transgene-flanking regions of multicopy potato transgenic
lines using the GOLD Genome Walking Kit (BIO S&T, Canada)
Cullen et al. 2011
Transgenic potato lines Copy number Junctions isolated (number of products analysed per
junction)
Left border Right border
Single event transformaton
Bkt 1- 1 2 2 2
Dxs - 36 2 ND 2
TPS6 2 1 2
TPS8 2 2 2
TPS16 2 ND 1
TPS42 2 2 ND
TPS4 3 3 ND
Co-transformed
CT - 184 2 4 2
CT - 3 3 4 2
CT - 88 3 3 3
Re-transformed
B9Zep-13 1 3 1
B9Zep-4 2 2 3 52

53. Fig. 10. Final-round PCR amplification products generated from the left border of two copy transgenic lines
of potato (TPS6 and TPS8) using the DNA Walking SpeedUp and GOLD genome kits and PCR products
loaded per lane.
A: Final round nested PCR products: lanes 2–5 ACP1 to ACP4 SpeedUp primers with TPS6, lanes 6–9 ACP1
to ACP4 SpeedUp primers with TPS8. B: Lanes 2–5 DRTA to DRTD APAgene primers with TPS6. lanes 6–9
DRTA to DRTD APAgene primers with TPS8.
Cullen et al. 2011
53

54. Fig. 11. Nucleotide sequence isolated from the right border of line B9ZEP-4 showing
DNA rearrangements in the vector backbone of pBIN19.
A short stretch of vector backbone sequence (36 bp) in light grey font showing 100%
homology to part of the tetA gene of pBIN19 (nucleotides 9,438–9,473).
54

55. Fig. 12. A stretch of vector backbone of 205 bp (underlined font) inserted in reverse
complement showing 100% homology to part of the kilA gene of pBIN19
(nucleotides 634–838).
55

56. Most efforts have been devoted to the identification of ‘regulatory
regions’, while a minor number of reports deal with the use of GW in
Gene identification, sequencing of BAC and YAC clones, cytoplasmic
male sterility (large modifications occurring in plant nuclear ⁄
mitochondrial genomes, which are at the basis of the cytoplasmic male
sterility phenotypic trait) and multigene families.
De novo sequencing
56

57. 57

58. 58

59. Fig. 15. PCR amplification of unknown flanking regions. Partially degenerate
primers randomly prime DNA synthesis and the Phi29 DNA polymerase
concurrently extends the primers as it displaces downstream DNA products
resulting in repeated replication of DNA fragments by a ‘‘hyperbanching”
mechanism of strand-displacement synthesis. Reddy et al. 200859

60. Fig. 16. The newly synthesized DNA fragments that carry a unique single-stranded walker adapter on
their 5` ends are used for subsequent PCR amplification of 5` or 3` flanking regions using locus-
specific, walker, and their corresponding nested primers in two rounds of PCR amplifications. The
solid and dashed lines are the two complementary strands of DNA.
The arrowheads represent the direction of DNA synthesis and the solid circles at the start of each line
represent the walker-adapter sequence. Reddy et al. 2008 60

61. Fig. 17. Amplified products after the second round of PCR. The different DNA
fragments amplified after two rounds of PCR-based genomic walk from four
(1, 2, 3,4) different walker-adapter primed whole genome-amplified
templates are on each lane. Reddy et al. 2008 61

62. Fig. 18. (B) Selected DNA fragments were cloned into TA-Topo cloning vector
and PCR-amplified using M13 (forward and reverse) primers. The names of the
promoters amplified are indicated on the top. Lane M represents the DNA size
standard markers (1-kb ladder). The numbers on the left represent the DNA
fragment length in kb. Reddy et al. 2008 62

63. Table 5. Details of the 5´ flanking sequence amplified in this study
63

64. Gene Organism GW approach ⁄ kit References
Po Shark Cassette PCR Fors et al. (1990)
TPA Man Suppression PCR Siebert et al. (1995)
PR-10 Parsley RAGE-GW Cormack and Somssich,
(1997)
Sucrose phosphate
synthase
Banana Single primer amplification Hermann et al. (2000)
Actin Sugarcane Single primer amplification Hermann et al. (2000)
Ribosomal protein Dunaliella tertiolecta Hermann et al. (2000)
Several cDNAs Pennisetum glaucum Mishra et al. (2002)
Gibberellin 20-
oxidase
Rice T-linker PCR Yuanxin et al. (2003)
Ascorbate
peroxidaseHsp70
P. glaucum High-throughput genome
walking
Reddy et al. (2008)
Gst Salicornia brachiata Reddy et al. (2008)
Lhcb1 family Spinach Leoni et al. (2010)
LRDEF Lily Straight walk Tsuchiya et al. (2009)
PGK1 Pichia ciferrii Template blocking PCR Bae and Sohn, (2010)
SUPERMAN-like Strawberry TVL-PCR Orcheski et al. (2010)
Table 6. Regulatory regions identified in eukaryotes by GW approaches
64

65. Multigene families
65

66. Objective: The method was used to study the spinach Lhcb1 family (encoding
the light harvesting complex protein Lhcb1), for which three cDNAs were
known.
Two additional genes and regulatory regions of the five members of the family
were identified.
66

67. Fig.19.(A) Electrophoretic characterization Of
genomic DNA digested with EcoRI restriction
enzyme and B. Southernblot Hybridization with a
Lhcb1.3 specific probe.
Leoni et al. 2010
67

68. 68

69.  Fig.20. Nucleotide sequenceof Lhcb1.4 ORF and its flanking regions. Nucleotides upstream
of the cDNA coding region have negative numbering.
 Coding sequence is reported in codons with corresponding amino-acids above.
 The first amino-acid of mature protein is underlined. Arrows indicate locations of primers
.Putative CAAT and TATA motifs areunderlined.
 GATA motifs, I-Box and a circadian expression element are boxed.
Leoni et al. 2010 69

70. Fig.21. Gel-shift assays of Lhcb1.4 regulatory elements.
•The 58bp probe contains the five regulatory elements detected upstream of the
Lhcb1.4 ORF
•The 31bp probe starts 5nt upstream of the I-box and also includes the GATA
Ib element. Leoni et al. 2010
70

71. Future line of work
• The extreme flexibility of GW strategies makes its application
possible in every standardly equipped research laboratory. In
addition, the possibility of merging GW strategies to next
generation sequencing approaches will undoubtedly
extend the future application of this by now basic technique of
molecular biology.
71

72. conclusionconclusion
It has largely contributed to advances in reverse genetic analysis,
and to the development of databases of mutants of many
eukaryotic genomes.
GW is particularly advantageous for the identification of specific
sequences in cases where whole genome sequencing projects have
not been undertaken.
It is noteworthy to observe that most of the different GW
strategies or improvements have been developed in the course of
de novo sequencing approaches
72

73. THANK YOU…
73