Genomic walking and sequencing by oligo-cassette mediated polymerase chain reaction

Rosenthal, André; Jones, Dan

doi:10.1093/nar/18.10.3095

Cited by 152 publications

(74 citation statements)

References 2 publications

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“…The third method used for isolating RBIP insertions is based on the genomic walking method (Figure 1, B and C; Rosenthal and Jones 1990;Siebert et al 1995). First, host sequences flanking the 39 ends of PDR1 insertion were used to design pairs of locus-specific nested primers (P 1 and P 2 ), oriented back toward the PDR1 insertion.…”

Section: Isolation Of Sequences Flanking Pdr1 Insertionsmentioning

confidence: 99%

Insertional Polymorphism and Antiquity ofPDR1Retrotransposon Insertions in Pisum Species

et al. 2005

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Sequences flanking 73 insertions of the retrotransposon PDR1 have been characterized, together with an additional 270 flanking regions from one side alone, from a diverse collection of Pisum germ plasm. Most of the identified flanking sequences are repetitious DNAs but more than expected (7%) lie within nuclear gene protein-coding regions. The approximate age of 52 of the PDR1 insertions has been determined by measuring sequence divergence among LTR pairs. These data show that PDR1 transpositions occurred within the last 5 MY, with a peak at 1-2.5 MYA. The insertional polymorphism of 68 insertions has been assessed across 47 selected Pisum accessions, representing the diversity of the genus. None of the insertions are fixed, showing that PDR1 insertions can persist in a polymorphic state for millions of years in Pisum. The insertional polymorphism data have been compared with the age estimations to ask what rules control the proliferation of PDR1 insertions in Pisum. Relatively recent insertions (, 1.5MYA) tend to be found in small subsets of the Pisum accessions set, ''middle-aged'' insertions (between 1.5 and 2.5 MYA) vary greatly in their occurrence, and older insertions (. 2.5 MYA) are mostly found in small subsets of Pisum. Finally, the average age estimate for PDR1 insertions, together with an existing data set for PDR1 retrotransposon SSAP markers, has been used to derive an estimate of the effective population size for Pisum of 7.5 3 10 5 .

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Section: Isolation Of Sequences Flanking Pdr1 Insertionsmentioning

confidence: 99%

Insertional Polymorphism and Antiquity ofPDR1Retrotransposon Insertions in Pisum Species

et al. 2005

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“…Importantly, both integrations could be detected in one reaction using a cocktail of the primers FP2 and FP4 (Figure 2b). In order to validate these results, we furthermore analyzed retroviral integration sites in these HT1080 cell line clones with a different method, an LM-PCR modified after Rosenthal and Jones 14 and Schmidt et al 7 After extensive studies with three different restriction enzymes to create a large variety of amplification permissive restriction fragment length polymorphisms, we found a total of three different retroviral integration sites in the two HT1080 cell line clones. These vector integrations were identical to those detected by two-step PCR with FP2, FP4 or FP5 as arbitrary primers (Table 1), thus confirming the reliability and sensitivity of the two-step PCR.…”

mentioning

confidence: 89%

Rapid detection of retroviral vector integration sites in colony-forming human peripheral blood progenitor cells using PCR with arbitrary primers

et al. 2003

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We have developed a highly sensitive polymerase chain reaction (PCR)-based technique termed two-step PCR, which uses arbitrary primers to identify proviral integration sites in retrovirally marked human colony-forming cells. The two-step PCR was established on cell line clones transduced with the SF1m retroviral vector and independently validated by demonstrating identical integration sites with ligationmediated PCR, a different technique requiring restriction enzyme digestion and adapter ligation for amplifying unknown DNA flanking the provirus. Two-step PCR was performed on peripheral blood progenitor cell (PBPC) colonies that contained as few as 75 cells, which was estimated by quantitative real-time PCR. We were able to amplify and directly sequence proviral integration sites in 35 % of PBPC colonies (25/72, five donors). Identity to the vector long-terminal repeat was confirmed and flanking DNA was found to match with human database sequences, reaffirming specificity. Two-step PCR is a valuable new tool for rapid analysis of genomic target sites for viral vectors, and will aid significantly in understanding clonal development of hematopoiesis and other cell types. Our protocol has the potential for general applicability as the arbitrary primers described here bind to genomic DNA and are thus independent of the vector backbone used.

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“…Several PCR-based approaches, such as inverse PCR (9) and adapter-mediated techniques (2,3,5,7,11), have emerged over the last decade. Although these techniques in many respects have simplified the cloning of flanking sequences, they still depend on a relatively labor-intensive restriction cutting for finding suitable restriction enzymes.…”

Section: Introductionmentioning

confidence: 99%

Restriction Cutting Independent Method for Cloning Genomic DNA Segments Outside the Boundaries of Known Sequences

1999

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We present a simple method for cloning genomic DNA segments outside the boundaries of known sequences, which is not dependent on restriction cutting or mapping. In the first step of the method, a library of single-stranded flanking sequences is generated by linear amplification with one primer in the known region. A homooligomeric cytosine tail is added to each of the single-stranded fragments by a terminal transferase catalyzed reaction. The tailed fragments are then amplified by PCR with a nested primer in the known region and a poly-guanine primer complementary to the cytosine tail in the unknown region. Finally, the different fragments are separated by cloning and characterized by sequencing. The method was used to clone both the upstream (5′) and the downstream (3′) genomic regions of an intron-interrupted tRNALeu(UAA) gene from three cyanobacteria belonging to the genus Microcystis.

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Genomic walking and sequencing by oligo-cassette mediated polymerase chain reaction

Cited by 152 publications

References 2 publications

Insertional Polymorphism and Antiquity ofPDR1Retrotransposon Insertions in Pisum Species

Insertional Polymorphism and Antiquity ofPDR1Retrotransposon Insertions in Pisum Species

Rapid detection of retroviral vector integration sites in colony-forming human peripheral blood progenitor cells using PCR with arbitrary primers

Restriction Cutting Independent Method for Cloning Genomic DNA Segments Outside the Boundaries of Known Sequences

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