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Most existing Tag-seq methods such as NlaIII-DGE (/ DpnII-DGE) or Super SAGE or 5′ SAGE methods use restriction enzymes and adapter ligation for cDNA tags.
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The massive undertaking to clone ORFs by the thousands cannot be easily achieved by conventional methods using restriction endonucleases.
In addition to sodium bisulfite treatment, digestion with methylation-sensitive restriction enzymes and several different methods using restriction enzymes combined with polymerase chain reaction 46 have been used for years.
Several methods using restriction enzymes have been developed for performing reduced representation sequencing for complex genomes (e.g. restriction-site-associated DNA sequencing (RAD-seq), reduced-representation libraries (RRL)) [ 27].
Therefore, for further identification of inserts we decided to exclusively use TAIL-PCR and no other methods such as inverse PCR or methods using restriction digestion and ligation [ 33].
While conventional cloning methods using restriction enzymes and polynucleotide ligase are adequate for most DNAs, fragments made by the polymerase chain reaction are difficult to clone because the amplifying DNA polymerase tends to add untemplated nucleotides to the 3'-termini of the amplified strands.
Secondly, the method uses restriction sites that may not be available in an amplified repeat unit.
As previously mentioned, the RAD method uses restriction enzymes as a complexity reduction strategy to reduce the sequenced portion of the genome anywhere from 0.01% to 10% [ 15].
Unlike conventional methods that use restriction enzymes or site-specific recombinases, PCR products could be seamlessly assembled without the need for specific sequences for ligation or site-specific recombination [ 25].
As a reference method, we used restriction fragment length polymorphism.
The clonal relation between isolates was investigated by PFGE methods using ApaI restriction enzyme.
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