^I just died for like 10 minutes. ohmygod. Why am I so funny?
This article talks about the development of a bacterial artificial chromosome recombineering procedure using galK-untranslated region for the mutation of diploid genes.
A common aim of researching genomics is to clone and analyze the entire genome of a species. For large eukaryotic genomes, it is easier when cloning vectors can accept larger chromosomal DNA inserts. Most plasmid and viral vectors can accommodate inserts that are a few thousands to tens of thousands of nucleotides in length. If a plasmid or viral vector has a DNA insert that is too large, it will have difficulty with DNA replication and is likely to suffer deletions in the insert. On the other hand, there is another type of cloning vector known as bacterial artificial chromosome (BAC). It can contain much larger inserted DNA fragments. BACs are derived from F factors, large plasmids. BACs are used in genomic research with the same use that other vectors are used for.
BAC recombineering using galK lets cloned DNA from E. coli to be modified without letting unwanted selectable markers (gene whose presence can allow organisms to grow under a certain set of conditions) in at the modification site. Certain genomes contain pairs of inverted repeat sequences that make it difficult to bring in mutations into duplicate genes using galKs selection method. A galK-UTR BAC recombineering procedure was created to mutate duplicate genes. This procedure blocks one copy of the target duplicate gene and allows the simple mutation of the other copy. Blocked copies are now able to be replaced with a UTR-specific primer pair.
In this experiment, mutant IR2 promoters that contained three Sp-1-binding motifs and a consensus TATA box were used in place of the two IR2 promotors in EHV-1 BAC. The results from this showed that there was a 4-fold increase of the expression level of the IR2 protein. This means that the galK-UTR method will prove as a useful tool in studies of herpesviruses.
This article talks about the development of a bacterial artificial chromosome recombineering procedure using galK-untranslated region for the mutation of diploid genes.
A common aim of researching genomics is to clone and analyze the entire genome of a species. For large eukaryotic genomes, it is easier when cloning vectors can accept larger chromosomal DNA inserts. Most plasmid and viral vectors can accommodate inserts that are a few thousands to tens of thousands of nucleotides in length. If a plasmid or viral vector has a DNA insert that is too large, it will have difficulty with DNA replication and is likely to suffer deletions in the insert. On the other hand, there is another type of cloning vector known as bacterial artificial chromosome (BAC). It can contain much larger inserted DNA fragments. BACs are derived from F factors, large plasmids. BACs are used in genomic research with the same use that other vectors are used for.
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| BAC doin' work. |
BAC recombineering using galK lets cloned DNA from E. coli to be modified without letting unwanted selectable markers (gene whose presence can allow organisms to grow under a certain set of conditions) in at the modification site. Certain genomes contain pairs of inverted repeat sequences that make it difficult to bring in mutations into duplicate genes using galKs selection method. A galK-UTR BAC recombineering procedure was created to mutate duplicate genes. This procedure blocks one copy of the target duplicate gene and allows the simple mutation of the other copy. Blocked copies are now able to be replaced with a UTR-specific primer pair.
In this experiment, mutant IR2 promoters that contained three Sp-1-binding motifs and a consensus TATA box were used in place of the two IR2 promotors in EHV-1 BAC. The results from this showed that there was a 4-fold increase of the expression level of the IR2 protein. This means that the galK-UTR method will prove as a useful tool in studies of herpesviruses.
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