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The use of multiple displacement amplification to amplify complex DNA libraries.

Fullwood MJ, Tan JJ, Ng PW, Chiu KP, Liu J, Wei CL, Ruan Y - Nucleic Acids Res. (2008)

Bottom Line: Complex libraries for genomic DNA and cDNA sequencing analyses are typically amplified using bacterial propagation.This process is time consuming, tedious and limits scaling up.However, MDA has no selection function for the removal of ligation multimers.

View Article: PubMed Central - PubMed

Affiliation: Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, Genome #02-01, Singapore.

ABSTRACT
Complex libraries for genomic DNA and cDNA sequencing analyses are typically amplified using bacterial propagation. To reduce biases, large numbers of colonies are plated and scraped from solid-surface agar. This process is time consuming, tedious and limits scaling up. At the same time, multiple displacement amplification (MDA) has been recently developed as a method for in vitro amplification of DNA. However, MDA has no selection function for the removal of ligation multimers. We developed a novel method of briefly introducing ligation reactions into bacteria to select single insert DNA clones followed by MDA to amplify. We applied these methods to a Gene Identification Signatures with Paired-End diTags (GIS-PET) library, which is a complex transcriptome library created by pairing short tags from the 5' and 3' ends of cDNA fragments together, and demonstrated that this selection and amplification strategy is unbiased and efficient.

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Related in: MedlinePlus

Schematic of the Selection-MDA approach. (A) Schematic showing the differences between the GIS-PET approach using bacterial propagation, and the GIS-PET approach using Selection-MDA. The Selection-MDA version allows for further amplification of the flcDNA library maxiprep by MDA, as well as amplification of the single-PET library solely by Selection-MDA without the need for tedious plating and scraping of large numbers of bacterial colonies from solid-surface agar. Approximate times required for steps that are different between different protocols are given in brackets. Comparing the steps between Selection-MDA and the bacterial propagation method, it is clear that Selection-MDA requires much less hands-on labor and time, and also, in terms of absolute time, is at least 4 h shorter. (B) Detailed schematic of the MDA protocol. FlcDNA maxiprep was cut with MmeI, self-ligated, and transformed into bacteria, which were recovered for 4 h. After this, cells were washed with media, plasmids were extracted. MDA was then performed, followed by enzymatic digestion, concatenation and then cloning and sequencing. We ran quality control aliquots of the reactions on PAGE gels after the plasmid purification. Clean plasmids of the correct size, 2800 bp, were obtained. After BamH1 digestion, 50 bp PETs were successfully recovered, as may be seen from the PAGE gel which shows a band of 50 bp (marked by a white box) separated from a high molecular weight smear from the plasmid backbone. PETs were successfully excised and concatenated, as may be seen from the smear from the concatemers, which was seen on a third PAGE gel. The concatemers were excised from the PAGE gel and prepared for subsequent cloning and sequencing.
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Figure 2: Schematic of the Selection-MDA approach. (A) Schematic showing the differences between the GIS-PET approach using bacterial propagation, and the GIS-PET approach using Selection-MDA. The Selection-MDA version allows for further amplification of the flcDNA library maxiprep by MDA, as well as amplification of the single-PET library solely by Selection-MDA without the need for tedious plating and scraping of large numbers of bacterial colonies from solid-surface agar. Approximate times required for steps that are different between different protocols are given in brackets. Comparing the steps between Selection-MDA and the bacterial propagation method, it is clear that Selection-MDA requires much less hands-on labor and time, and also, in terms of absolute time, is at least 4 h shorter. (B) Detailed schematic of the MDA protocol. FlcDNA maxiprep was cut with MmeI, self-ligated, and transformed into bacteria, which were recovered for 4 h. After this, cells were washed with media, plasmids were extracted. MDA was then performed, followed by enzymatic digestion, concatenation and then cloning and sequencing. We ran quality control aliquots of the reactions on PAGE gels after the plasmid purification. Clean plasmids of the correct size, 2800 bp, were obtained. After BamH1 digestion, 50 bp PETs were successfully recovered, as may be seen from the PAGE gel which shows a band of 50 bp (marked by a white box) separated from a high molecular weight smear from the plasmid backbone. PETs were successfully excised and concatenated, as may be seen from the smear from the concatemers, which was seen on a third PAGE gel. The concatemers were excised from the PAGE gel and prepared for subsequent cloning and sequencing.

Mentions: To construct the MDA-amplified library using the new Selection-MDA protocol (Figure 2), we took an aliquot of 8 ng of maxiprep from the GIS-PET full-length cDNA library and added it to 50 μl of Templiphi 500 sample buffer (GE Healthcare). The sample was denatured at 95°C for 3 min, and then cooled to 4°C. 2 μl of Templiphi 500 enzyme mix (GE Healthcare) was added to 50 μl Templiphi sample buffer on ice, and the mixture was then added to the 50 μl sample buffer with denatured template. The reaction was incubated at 30°C for 18 h, and then heat inactivated at 65°C for 10 min. The material was quantitated with Picogreen Fluorimetry (Invitrogen), and an MmeI (New England Biolabs) digestion was performed following the Single PET construction method as described (12). 800 ng of self-ligation reaction was purified to remove salts before electroporation by phenol/chloroform isopropanol precipitation as described (12). The pellet was resuspended in 5 μl of Elution Buffer (Qiagen). The entire ligation mix was transformed into 50 μl of Top10 E. coli electrocompetent cells (Invitrogen) and recovered in 1 ml of Lucigen Recovery Medium (Lucigen) with shaking at 37°C for 4 h. Because recovery was for only 4 h, the bacteria would not have multiplied sufficiently so as to compete with each other; hence the library should contain no size bias. To monitor bacterial growth, the optical density at 600 nm (OD600) of aliquots were taken at various time points by an ND-1000 spectrophotometer (Nanodrop). Cells were spun down at 10 000 g for 5 min and washed twice with 750 μl of Lucigen Recovery Medium to remove free-floating DNA that was not introduced into the cells. Next, plasmids were extracted by performing Miniprep (Qiagen). 40 μl of elution buffer was used for the elution, and the DNA was quantitated with Picogreen fluorimetry. 1 μl was run on a PAGE gel to check that plasmids were prepared correctly (Figure 2B, ‘purified plasmids’). Plasmid-safe DNAse (Epicenter) treatment was then performed to remove any linear species, such as bacterial genomic DNA, that might be present. Phenol/chloroform ethanol precipitation was then performed and pellets were resuspended in 20 μl of Elution Buffer (Qiagen). MDA was performed on aliquots of 8 ng of material as described earlier. The material was quantitated with Picogreen Fluorimetry, and digested with BamHI (New England Biolabs) according to the manufacturer's protocols. The PETs were PAGE gel-purified (Figure 2B, ‘50 bp ditags obtained after BamHI digest’), then cloned, concatenated (Figure 2B, ‘concatenated BamHI-cut PETs’), partially digested with BamHI, cloned into BamHI-cut pZErO-1 vectors (Invitrogen), and prepared for sequencing as described (12). Ten plates of 384 colonies consisting of concatenated PETs were sequenced as a GIS-PET library, SHE002. A more detailed protocol is provided in the Supplementary Data.Figure 2.


The use of multiple displacement amplification to amplify complex DNA libraries.

Fullwood MJ, Tan JJ, Ng PW, Chiu KP, Liu J, Wei CL, Ruan Y - Nucleic Acids Res. (2008)

Schematic of the Selection-MDA approach. (A) Schematic showing the differences between the GIS-PET approach using bacterial propagation, and the GIS-PET approach using Selection-MDA. The Selection-MDA version allows for further amplification of the flcDNA library maxiprep by MDA, as well as amplification of the single-PET library solely by Selection-MDA without the need for tedious plating and scraping of large numbers of bacterial colonies from solid-surface agar. Approximate times required for steps that are different between different protocols are given in brackets. Comparing the steps between Selection-MDA and the bacterial propagation method, it is clear that Selection-MDA requires much less hands-on labor and time, and also, in terms of absolute time, is at least 4 h shorter. (B) Detailed schematic of the MDA protocol. FlcDNA maxiprep was cut with MmeI, self-ligated, and transformed into bacteria, which were recovered for 4 h. After this, cells were washed with media, plasmids were extracted. MDA was then performed, followed by enzymatic digestion, concatenation and then cloning and sequencing. We ran quality control aliquots of the reactions on PAGE gels after the plasmid purification. Clean plasmids of the correct size, 2800 bp, were obtained. After BamH1 digestion, 50 bp PETs were successfully recovered, as may be seen from the PAGE gel which shows a band of 50 bp (marked by a white box) separated from a high molecular weight smear from the plasmid backbone. PETs were successfully excised and concatenated, as may be seen from the smear from the concatemers, which was seen on a third PAGE gel. The concatemers were excised from the PAGE gel and prepared for subsequent cloning and sequencing.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2275127&req=5

Figure 2: Schematic of the Selection-MDA approach. (A) Schematic showing the differences between the GIS-PET approach using bacterial propagation, and the GIS-PET approach using Selection-MDA. The Selection-MDA version allows for further amplification of the flcDNA library maxiprep by MDA, as well as amplification of the single-PET library solely by Selection-MDA without the need for tedious plating and scraping of large numbers of bacterial colonies from solid-surface agar. Approximate times required for steps that are different between different protocols are given in brackets. Comparing the steps between Selection-MDA and the bacterial propagation method, it is clear that Selection-MDA requires much less hands-on labor and time, and also, in terms of absolute time, is at least 4 h shorter. (B) Detailed schematic of the MDA protocol. FlcDNA maxiprep was cut with MmeI, self-ligated, and transformed into bacteria, which were recovered for 4 h. After this, cells were washed with media, plasmids were extracted. MDA was then performed, followed by enzymatic digestion, concatenation and then cloning and sequencing. We ran quality control aliquots of the reactions on PAGE gels after the plasmid purification. Clean plasmids of the correct size, 2800 bp, were obtained. After BamH1 digestion, 50 bp PETs were successfully recovered, as may be seen from the PAGE gel which shows a band of 50 bp (marked by a white box) separated from a high molecular weight smear from the plasmid backbone. PETs were successfully excised and concatenated, as may be seen from the smear from the concatemers, which was seen on a third PAGE gel. The concatemers were excised from the PAGE gel and prepared for subsequent cloning and sequencing.
Mentions: To construct the MDA-amplified library using the new Selection-MDA protocol (Figure 2), we took an aliquot of 8 ng of maxiprep from the GIS-PET full-length cDNA library and added it to 50 μl of Templiphi 500 sample buffer (GE Healthcare). The sample was denatured at 95°C for 3 min, and then cooled to 4°C. 2 μl of Templiphi 500 enzyme mix (GE Healthcare) was added to 50 μl Templiphi sample buffer on ice, and the mixture was then added to the 50 μl sample buffer with denatured template. The reaction was incubated at 30°C for 18 h, and then heat inactivated at 65°C for 10 min. The material was quantitated with Picogreen Fluorimetry (Invitrogen), and an MmeI (New England Biolabs) digestion was performed following the Single PET construction method as described (12). 800 ng of self-ligation reaction was purified to remove salts before electroporation by phenol/chloroform isopropanol precipitation as described (12). The pellet was resuspended in 5 μl of Elution Buffer (Qiagen). The entire ligation mix was transformed into 50 μl of Top10 E. coli electrocompetent cells (Invitrogen) and recovered in 1 ml of Lucigen Recovery Medium (Lucigen) with shaking at 37°C for 4 h. Because recovery was for only 4 h, the bacteria would not have multiplied sufficiently so as to compete with each other; hence the library should contain no size bias. To monitor bacterial growth, the optical density at 600 nm (OD600) of aliquots were taken at various time points by an ND-1000 spectrophotometer (Nanodrop). Cells were spun down at 10 000 g for 5 min and washed twice with 750 μl of Lucigen Recovery Medium to remove free-floating DNA that was not introduced into the cells. Next, plasmids were extracted by performing Miniprep (Qiagen). 40 μl of elution buffer was used for the elution, and the DNA was quantitated with Picogreen fluorimetry. 1 μl was run on a PAGE gel to check that plasmids were prepared correctly (Figure 2B, ‘purified plasmids’). Plasmid-safe DNAse (Epicenter) treatment was then performed to remove any linear species, such as bacterial genomic DNA, that might be present. Phenol/chloroform ethanol precipitation was then performed and pellets were resuspended in 20 μl of Elution Buffer (Qiagen). MDA was performed on aliquots of 8 ng of material as described earlier. The material was quantitated with Picogreen Fluorimetry, and digested with BamHI (New England Biolabs) according to the manufacturer's protocols. The PETs were PAGE gel-purified (Figure 2B, ‘50 bp ditags obtained after BamHI digest’), then cloned, concatenated (Figure 2B, ‘concatenated BamHI-cut PETs’), partially digested with BamHI, cloned into BamHI-cut pZErO-1 vectors (Invitrogen), and prepared for sequencing as described (12). Ten plates of 384 colonies consisting of concatenated PETs were sequenced as a GIS-PET library, SHE002. A more detailed protocol is provided in the Supplementary Data.Figure 2.

Bottom Line: Complex libraries for genomic DNA and cDNA sequencing analyses are typically amplified using bacterial propagation.This process is time consuming, tedious and limits scaling up.However, MDA has no selection function for the removal of ligation multimers.

View Article: PubMed Central - PubMed

Affiliation: Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, Genome #02-01, Singapore.

ABSTRACT
Complex libraries for genomic DNA and cDNA sequencing analyses are typically amplified using bacterial propagation. To reduce biases, large numbers of colonies are plated and scraped from solid-surface agar. This process is time consuming, tedious and limits scaling up. At the same time, multiple displacement amplification (MDA) has been recently developed as a method for in vitro amplification of DNA. However, MDA has no selection function for the removal of ligation multimers. We developed a novel method of briefly introducing ligation reactions into bacteria to select single insert DNA clones followed by MDA to amplify. We applied these methods to a Gene Identification Signatures with Paired-End diTags (GIS-PET) library, which is a complex transcriptome library created by pairing short tags from the 5' and 3' ends of cDNA fragments together, and demonstrated that this selection and amplification strategy is unbiased and efficient.

Show MeSH
Related in: MedlinePlus