Welcome to the web supplement to:

"Development and validation of a T7 based linear amplification for genomic DNA", BMC Genomics 2003 May 9, 4:19

and

"Chapter 7: DNA linear amplification", Whole Genome Amplification: Methods Express, Hughes S. and Lasken R., eds. Scion Publishing Ltd., Bloxham, Oxfordshire, United Kingdom. (PDF here)

FAQ - Frequently Asked Questions

Access the FAQ, containing general information, troubleshooting tips, and recommended controls on this DNA linear amplification method.

(last updated 3/29/2006 by Chih Long Liu)

Download Data

Microarray Data, GenePix Report (GPR) format
These files constitute the unprocessed microarray data as acquired with GenePix 4.0 upon the initial image acquisition of the microarrays. Each file is plain-text readable but is best opened either under GenePix or a spreadsheet program such as Microsoft Excel.
File Size Description
Direct Label
Replicate 1
Replicate 2
Replicate 3 		1.8 MB each GPR files for Alu/Rsa yeast ORF array, direct labeled sample
IVT-amplified
Replicate 1
Replicate 2
Replicate 3 		1.8 MB each GPR files for Alu/Rsa yeast ORF array, IVT-amplified samples
R-PCR amplified
Replicate 1
Replicate 2
Replicate 3 		1.8 MB each GPR files for Alu/Rsa yeast ORF array, IVT-amplified samples
IVT-amplified
5 ng
 1.8 MB GPR files for Alu/Rsa yeast ORF array. This file corresponds to a single sample that was amplified from 5 ng of starting material.
Additional files
These are additional files that supplement this work. These include TreeView files used to generate Figure 5 as well as updated protocols and other helpful information.
File Size Description
Figure 5
TreeView Files
GTR, ATR, CDT 		688 kb
total		 TreeView files used to generate Figure 5. Use this to browse the Figure 5 dataset locally on your computer. You can find TreeView here.
TreeView files:
GTR = Gene dendrogram
ATR = Array dendrogram
CDT = Clustered output
Note: the CDT file contains the normalized, log2 ratio data of the dataset. Open this file in a spreadsheet program if you wish to browse the data.
IVT amplification protocol
PDF Ver. 3.13 (4-21-03)
PDF Ver. 3.21 (5-28-05)
 55-60 kb Detailed lab manual-format protocol of the DNA T7 IVT amplification method. The BMC Genomics paper is based on Version 3.13; the WGA Methods Chapter (PDF) is based on Version 3.21 and includes a number of updates and additional optimizations. It is recommended that Version 3.21 of the protocol be followed, particularly since it incorporates a change in a buffer provided by a supplier and also contains a detailed table of reaction volumes for the second strand synthesis step.
Reverse transcription and labeling protocol
PDF (5/28/05)
 35 kb Detailed lab manual-format protocol of the reverse transcription labeling protocol in use at the Schreiber Lab for ChIP-chip experiments. This protocol is slightly modified from the version available on www.microarrays.org. This protocol uses the amino-allyl method for coupling fluorescent dyes to the probe and is the recommended RT protocol for use with TLAD. Users wishing to use the direct incorporation method may do so as well. Users who wish to perform RNA hybridization can substitute in a ratio of modified nucleotides during the IVT step.

Additional Recommendations

For new users of this protocol, it is highly recommended that the following two positive controls be run:

Whole-protocol amplification control.

This control will enable the user to distinguish sample-specific problems from protocol implementation issues. Here, the starting material is 50 ng of blunt-ended PCR product in the 100-1000 bp range (preferably around 200-500 bp). If this protocol is used as part of a ChIP-chip experiment, and if troubleshooting RNase A handing and CIP treatment, the PCR product can be subject to those treatments as well, though in practice, it is usually not necessary. Yields should typically range from 30-60 ug (with a maximum observed of about 80-100 ug), depending on the size of the PCR product, protocol implementation, and quality of the reagents used (particularly nucleotides used for IVT, which are highly sensitive to freeze-thaw cycles).

IVT control.

This control will enable the user to distinguish sample-specific problems from IVT-specific problems (such as RNase contamination). Here, the IVT starting material is 250 ng of the pTRI-Xef linearized plasmid provided with the Ambion IVT kit. If not using the kit, an appropriate amount of a dsDNA template that contains the pT7 promoter, with prior history of use as a successful T7 RNA polymerase template, should be used. Yields should typically range from 100-140 ug, limited by exhaustion of the nucleotides in the reaction mixture and the rated 100 ug binding capacity of the Qiagen RNeasy column. Any yields sufficiently less than this may suggest RNase contamination, evaporation of reaction mixture during the long incubation period (due to the small reaction volume), or poor reagent quality (particularly with the nucleotides). Visualization on a non-denaturing agarose gel should yield two intense bands ~1.8 kb.

Extensive details of this method appear in Chapter 7 of the laboratory manual, Whole Genome Amplification: Methods Express. The manual has recently been published (Oct. 2005), and you can find our chapter (PDF format) here. We highly recommend that you read this chapter because of the extensive information available there. (The WGA Methods Express contains a good number of other DNA amplification methods, in case you're interested.) In the meantime, check out the FAQ.

Last updated by Chih Long Liu on 3/29/2006.