A simplified method of constructing infectious clones of begomovirus employing limited restriction enzyme digestion of products of rolling circle amplification

2008 ◽  
Vol 147 (2) ◽  
pp. 355-359 ◽  
Author(s):  
Chia-Ying Wu ◽  
Yi-Chin Lai ◽  
Na-Sheng Lin ◽  
Yau-Heiu Hsu ◽  
Hsin-Tzu Tsai ◽  
...  
Keyword(s):  
Restriction Enzyme ◽  
Rolling Circle Amplification ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion ◽  
Rolling Circle ◽  
Infectious Clones ◽  
Simplified Method

scholarly journals A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification

2004 ◽  
Vol 78 (10) ◽  
pp. 4993-4998 ◽  
Author(s):  
Annabel Rector ◽  
Ruth Tachezy ◽  
Marc Van Ranst
Keyword(s):  
Restriction Enzyme ◽  
Rolling Circle Amplification ◽  
Enzyme Analysis ◽  
Amplification Efficiency ◽  
Sequence Information ◽  
Restriction Enzyme Digestion ◽  
Hpv 16 ◽  
Circular Dna ◽  
Rolling Circle ◽  
Papillomavirus Dna

ABSTRACT The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with φ29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 × 104-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information.

The stability of maize nuclei to restriction enzyme digestion differs with transcriptional activity

Caryologia ◽  
1993 ◽  
Vol 46 (1) ◽  
pp. 63-69 ◽  
Author(s):  
Valeria Mirkova ◽  
Maria Ivanchenko ◽  
Lubomir Stoilov ◽  
Jordanka Zlatanova
Keyword(s):  
Restriction Enzyme ◽  
Transcriptional Activity ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion ◽  
The Stability

Identification of the CD32/FcγRIIc-Q13/STP13 polymorphism using an allele-specific restriction enzyme digestion assay

2001 ◽  
Vol 258 (1-2) ◽  
pp. 85-95 ◽  
Author(s):  
D Metes ◽  
A.A Gambotto ◽  
J Nellis ◽  
A Ruscin ◽  
A.M Stewart-Akers ◽  
...  
Keyword(s):  
Restriction Enzyme ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion ◽  
Allele Specific ◽  
Specific Restriction

Isolation of large terminal sequences of BAC inserts based on double-restriction-enzyme digestion followed by anchored PCR

Genome ◽  
2000 ◽  
Vol 43 (2) ◽  
pp. 412-415 ◽  
Author(s):  
Zhong-Nan Yang ◽  
T Erik Mirkov
Keyword(s):  
Restriction Enzyme ◽  
Genomic Dna ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion ◽  
Artificial Chromosomes ◽  
Bac Clones ◽  
Anchored Pcr ◽  
Pcr Products ◽  
Left And Right ◽  
The Right

Isolation of the terminal portions of genomic DNA cloned in bacterial artificial chromosomes (BACs) is an important step in map-based cloning, and several methods have been developed. Here, we present a new method based on double-restriction-enzyme digestion followed by anchored PCR. BAC DNA was digested with two enzymes: NotI and one of four enzymes (EcoRV, HpaI, StuI, or XmnI) that produce blunt termini. After dephosphorylation, these digestions were ligated to NotI- and EcoRV-digested pMSK, a new cloning vector developed in this work that is derived from pBluescript SK(+). PCR products representing the left- and right-terminal sequences of BAC inserts were obtained using a primer complementary to pMSK and a primer complementary to sequences in either the left arm or the right arm of the BAC vector pBeloBAC11. We have tested this method with 15 different BAC clones, and PCR products representing both the left- and right-terminal sequences have been obtained from all 15 BAC clones. This method is simple, fast, reproducible, and uses the same set of primers for any restriction enzyme used. With some modifications, it can also be used for isolating the terminal portions of genomic DNA cloned in yeast artificial chromosomes and P1-derived artificial chromosomes. Key words: BAC, anchored PCR, terminal sequence isolation, chromosome walk.

Resistance of Tumor-Derived DNA to Restriction Enzyme Digestion

1990 ◽  
Vol 8 (2) ◽  
pp. 169-172
Author(s):  
Joan Lee Parkes ◽  
Frank C. Hubbard ◽  
Arthur Penn
Keyword(s):  
Restriction Enzyme ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion
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