Dna Shuffling
Dna Shuffling - Related Videos
21:55
Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling
0 Views •
Cited by 35 •
2012
We demonstrate the basic technique to molecularly engineer and evolve synthetic Adeno-associated viral (AAV) gene therapy vectors via DNA family shuffling. Moreover, we provide general guidelines and representative examples for selection and analysis of individual chimeric capsids with enhanced properties on target cells in culture or in mice.
09:31
Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites
0 Views •
Cited by 56 •
2016
We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.
10:20
Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase
0 Views •
Cited by 2 •
2008
Current HIV-1 strategies act to suppress the viral life cycle but do not effectively eradicate infection. Here, we demonstrate that an engineered recombinase can efficiently excise integrated HIV-1 proviral DNA from the genome of infected cells.
14:57
DNA Stable-Isotope Probing (DNA-SIP)
0 Views •
Cited by 70 •
2010
DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.
02:02
DNA Topoisomerases
0 Views •
2020
Topoisomerases are enzymes that relax overwound DNA molecules during various cell processes, including DNA replication and transcription. These enzymes regulate positive and negative DNA supercoiling without changing the nucleotide sequence. DNA overwinding in a clockwise direction results in positively supercoiled DNA, whereas underwinding in a counterclockwise direction produces negatively supercoiled DNA. Types and Mechanism of action Topoisomerases are divided into two main types. Type I...
FAQs
DNase I fragmentation breaks related genes into pieces that retain stretches of sequence similarity with one another. During primerless PCR, an overlapping fragment can align to a complementary region from another parent and extend across that junction. Repeated rounds of this overlap-guided assembly produce full-length molecules containing combinations of parental segments, creating chimeric gene variants for later testing.
Unlike a primer-directed amplification step, primerless PCR relies on the fragments themselves as templates and extending partners. Overlap between fragments supplies the alignment needed for synthesis, so sequences from different parental genes can become joined without designing a separate primer for every crossover. This makes the assembly stage central to recovering diverse, full-length recombinant genes.
Related parental sequences provide homologous regions that allow fragments to recognize and join one another during assembly. Their shared sequence features make recombination possible across different parent combinations. The resulting library can connect sequence changes with protein properties, supporting studies of how genetic variation influences activity, stability, specificity, or expression.
A practical workflow begins with selecting related homologous genes, fragmenting them, and allowing the pieces to reassemble through primerless PCR. An amplification step then recovers complete chimeric genes rather than leaving only short fragments. Researchers introduce these gene variants into suitable host cells, where the encoded proteins can be examined in a screening or selection stage.
Screening or selection connects each recovered gene variant with the performance of its encoded protein. Researchers can look for improved activity, stability, specificity, or expression, depending on the experimental goal. Comparing successful variants with their parental sequences also supports functional studies of sequence-property relationships, helping identify which recombined genetic changes accompany a desired protein characteristic.
It supplies a way to explore combinations of sequence features from related genes rather than evaluating only the original parental forms. In biotechnology, this supports directed evolution and enzyme development by generating variants for screening. In biology, the same strategy helps investigate how altered gene sequences relate to protein function, including changes in activity, stability, specificity, or expression.