Limits...
Two-dimensional DNA displays for comparisons of bacterial genomes.

Malloff C, Dullaghan E, Li A, Stokes R, Fernandez R, Lam W - Biol Proced Online (2003)

Bottom Line: BCGH relies on performing 2DBGD on a pooled sample of genomic DNA from 2 strains to be compared and subsequently hybridizing the resulting 2DBGD blot separately with DNA from each individual strain.Unique spots (hybridization signals) represent foreign DNA.The identification of novel DNA is easily achieved by excising the DNA from a dried gel followed by subsequent cloning and sequencing. 2DBGD and BCGH thus represent novel high resolution genome scanning techniques for directly identifying altered and/or acquired DNA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Pathology and Laboratory Medicine.

ABSTRACT
We have developed two whole genome-scanning techniques to aid in the discovery of polymorphisms as well as horizontally acquired genes in prokaryotic organisms. First, two-dimensional bacterial genomic display (2DBGD) was developed using restriction enzyme fragmentation to separate genomic DNA based on size, and then employing denaturing gradient gel electrophoresis (DGGE) in the second dimension to exploit differences in sequence composition. This technique was used to generate high-resolution displays that enable the direct comparison of > 800 genomic fragments simultaneously and can be adapted for the high-throughput comparison of bacterial genomes. 2DBGDs are capable of detecting acquired and altered DNA, however, only in very closely related strains. If used to compare more distantly related strains (e.g. different species within a genus) numerous small changes (i.e. small deletions and point mutations) unrelated to the interesting phenotype, would encumber the comparison of 2DBGDs. For this reason a second method, bacterial comparative genomic hybridization (BCGH), was developed to directly compare bacterial genomes to identify gain or loss of genomic DNA. BCGH relies on performing 2DBGD on a pooled sample of genomic DNA from 2 strains to be compared and subsequently hybridizing the resulting 2DBGD blot separately with DNA from each individual strain. Unique spots (hybridization signals) represent foreign DNA. The identification of novel DNA is easily achieved by excising the DNA from a dried gel followed by subsequent cloning and sequencing. 2DBGD and BCGH thus represent novel high resolution genome scanning techniques for directly identifying altered and/or acquired DNA.

No MeSH data available.


Related in: MedlinePlus

Schematic outlining Bacterial Comparative Genomic Hybridization. (a) Samples to be compared are digested with the same frequent cutting enzyme(s) and combined. (b) The combined sample is then size separated on a first dimension polyacrylamide gel. (c) The first dimension gel lane is then cut from the gel and transferred to the top of a denaturing gradient polyacrylamide gel. (d) Sample fragments are then resolved in the second dimension based on melting characteristics. (e) The gel is then washed and the DNA electroblotted to a nylon membrane. (f,g) The nylon membrane is then alternatively hybridized with radiolabelled genomic DNA probe (asterisk) of generated from the comparison samples.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC162171&req=5

Figure 2: Schematic outlining Bacterial Comparative Genomic Hybridization. (a) Samples to be compared are digested with the same frequent cutting enzyme(s) and combined. (b) The combined sample is then size separated on a first dimension polyacrylamide gel. (c) The first dimension gel lane is then cut from the gel and transferred to the top of a denaturing gradient polyacrylamide gel. (d) Sample fragments are then resolved in the second dimension based on melting characteristics. (e) The gel is then washed and the DNA electroblotted to a nylon membrane. (f,g) The nylon membrane is then alternatively hybridized with radiolabelled genomic DNA probe (asterisk) of generated from the comparison samples.

Mentions: As previously described (14), Bacterial Comparative Genomic Hybridization (BCGH) is outlined in Figure 2A-G.


Two-dimensional DNA displays for comparisons of bacterial genomes.

Malloff C, Dullaghan E, Li A, Stokes R, Fernandez R, Lam W - Biol Proced Online (2003)

Schematic outlining Bacterial Comparative Genomic Hybridization. (a) Samples to be compared are digested with the same frequent cutting enzyme(s) and combined. (b) The combined sample is then size separated on a first dimension polyacrylamide gel. (c) The first dimension gel lane is then cut from the gel and transferred to the top of a denaturing gradient polyacrylamide gel. (d) Sample fragments are then resolved in the second dimension based on melting characteristics. (e) The gel is then washed and the DNA electroblotted to a nylon membrane. (f,g) The nylon membrane is then alternatively hybridized with radiolabelled genomic DNA probe (asterisk) of generated from the comparison samples.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC162171&req=5

Figure 2: Schematic outlining Bacterial Comparative Genomic Hybridization. (a) Samples to be compared are digested with the same frequent cutting enzyme(s) and combined. (b) The combined sample is then size separated on a first dimension polyacrylamide gel. (c) The first dimension gel lane is then cut from the gel and transferred to the top of a denaturing gradient polyacrylamide gel. (d) Sample fragments are then resolved in the second dimension based on melting characteristics. (e) The gel is then washed and the DNA electroblotted to a nylon membrane. (f,g) The nylon membrane is then alternatively hybridized with radiolabelled genomic DNA probe (asterisk) of generated from the comparison samples.
Mentions: As previously described (14), Bacterial Comparative Genomic Hybridization (BCGH) is outlined in Figure 2A-G.

Bottom Line: BCGH relies on performing 2DBGD on a pooled sample of genomic DNA from 2 strains to be compared and subsequently hybridizing the resulting 2DBGD blot separately with DNA from each individual strain.Unique spots (hybridization signals) represent foreign DNA.The identification of novel DNA is easily achieved by excising the DNA from a dried gel followed by subsequent cloning and sequencing. 2DBGD and BCGH thus represent novel high resolution genome scanning techniques for directly identifying altered and/or acquired DNA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Pathology and Laboratory Medicine.

ABSTRACT
We have developed two whole genome-scanning techniques to aid in the discovery of polymorphisms as well as horizontally acquired genes in prokaryotic organisms. First, two-dimensional bacterial genomic display (2DBGD) was developed using restriction enzyme fragmentation to separate genomic DNA based on size, and then employing denaturing gradient gel electrophoresis (DGGE) in the second dimension to exploit differences in sequence composition. This technique was used to generate high-resolution displays that enable the direct comparison of > 800 genomic fragments simultaneously and can be adapted for the high-throughput comparison of bacterial genomes. 2DBGDs are capable of detecting acquired and altered DNA, however, only in very closely related strains. If used to compare more distantly related strains (e.g. different species within a genus) numerous small changes (i.e. small deletions and point mutations) unrelated to the interesting phenotype, would encumber the comparison of 2DBGDs. For this reason a second method, bacterial comparative genomic hybridization (BCGH), was developed to directly compare bacterial genomes to identify gain or loss of genomic DNA. BCGH relies on performing 2DBGD on a pooled sample of genomic DNA from 2 strains to be compared and subsequently hybridizing the resulting 2DBGD blot separately with DNA from each individual strain. Unique spots (hybridization signals) represent foreign DNA. The identification of novel DNA is easily achieved by excising the DNA from a dried gel followed by subsequent cloning and sequencing. 2DBGD and BCGH thus represent novel high resolution genome scanning techniques for directly identifying altered and/or acquired DNA.

No MeSH data available.


Related in: MedlinePlus