APPLICATION NOTE Sizing of Large DNA Fragments
Sizing of Large DNA Fragments
Generated by BAC Fingerprinting on Capillary Electrophoresis System
6000
5000
4000
3000
2000
1000
0
1,330
3,330
5,330
7,330
9,930
11,330
11 330
13,330
13 330
15 330
15,330
Landmark Peaks
6000 15,330
5000
4000
3000
2000
1000
0
5,420 5,820 6,220 6,620 7,020
7,420
7,820
8,220 8,620 9,020 9,420
Figure 1. Electropherogram of the GeneScan ™ 1200 LIZ ® Size Standard. Expanded range shown between 300 - 600 bp provides a clear illustration of the
landmark fragments.
Introduction
BAC fingerprinting provides an efficient
and cost-effective method of characterizing large genomic fragment libraries for
genome sequencing, positional cloning,
and physical mapping efforts.
Restriction endonuclease digestion of
BAC clones followed by fluorescent dye
labeling can be used to generate a profile
or fingerprint. Overlap between fingerprints are subsequently used to assemble
contiguous sequences (contigs) in the
construction of whole-genome physical
maps. Physical maps are important resources for genome sequencing efforts,
positional cloning, comparative genomics,
and to determine the size and structure
of genomes.
In this application note, the GeneScan
1200 LIZ Size Standard (a high-density
size standard for sizing DNA fragments
up to 1200 bp with a high degree of
precision) is used along with the
SNaPshot® Multiplex System for BAC
fingerprinting of wheat DNA fragments
analyzed by capillary electrophoresis
on an Applied Biosystems 3730xl DNA
Analyzer.
Overview of the GeneScan 1200 LIZ
Size Standard
The GeneScan 1200 LIZ Size Standard
is designed for larger DNA fragment
sizing applications, and leverages
Applied Biosystems proprietary
5-dye technology.
•
Fragments from 20—1200 bp enable
analysis of a wide range of DNA
fragment lengths
•
High fragment density (68 fragments)
yields greater sizing precision
•
Landmark fragments included for easy
peak pattern identification during
data analysis
•
•
Size standard fragments labeled with
LIZ dye for 5-dye fragment analysis
applications, allowing 33% greater
multiplexing throughput than
4-dye applications
Ideal for BAC fingerprinting, AFLP,
T-RFLP, VNTR, STR, and many other
DNA fragment analysis applications
Overview of the SNaPshot
Multiplex System:
The kit offers a one-tube, single-base
extension/termination reagent for
labeling. SNaPshot chemistry is based
on the dideoxy single-base extension
of an unlabeled oligonuceotide primer
or primers. For BAC fingerprinting, the
chemistry extends from the 3’ recessed
end of restriction fragments, with the
5’ overhang serving as template. Four
fluorescent dyes are used to distinguish
each nucleotide.
The BAC Fingerprinting Workflow
After the desired BACs are obtained,
sample preparation begins with the
following steps:
1. Selective growth of BAC
containing bacteria
2. BAC DNA purification
3. Restriction endonuclease digestion
4. Fragment end-labeling using the
SNaPshot® Multiplex Kit
5. Post-extension clean-up of the
labeled fragments
The workflow begins with the growth
and isolation of the BAC DNA. This is
followed by restriction endonuclease
digestion of the BAC clones with several
different enzymes. The fragments are
then labeled with fluorescent dye-labeled
primers included in the SNaPshot kit. The
dye-labeled primers are bound to the BAC
fragments based on the overhangs left by
the restriction enzymes (see Table 1).
The pool of dye-labeled DNA fragments
are analyzed on a capillary electrophoresis
system such as the Applied Biosystems
3730xl. Because the technique can
generate large DNA fragments, the
GeneScan 1200 LIZ Size Standard is
chosen for analysis with the dye labeled
BAC fragments. When analyzed by
electrophoresis, the dye-labeled BAC
fragments generate a fragment “fingerprint.” Overlap between fingerprints are
used to assemble contigs for the construction of whole-genome physical maps.
These physical maps serve as important
resources for genome sequencing efforts,
positional cloning, comparative genomics,
and to determine the size and structure
of genomes.
Precision Sizing of Large Wheat
DNA Fragments
In this study, control wheat BAC samples
were digested, labeled, and analyzed on
the Applied Biosystems 3730xl Genetic
Analyzer using POP-7™ polymer, a 50-cm
capillary array and using GeneMapper®
v4.0 Software. Sizing precision (allele size
standard deviation) per run was calculated
from 276 samples. (See Figures 2 and 3 for
analysis results)
TABLE 1. RESTRICTION ENDONUCLEASES IN BAC FINGERPRINTING
Enzyme
Site
ddNTP
Dye
Color
EcoRI
G^AATTC
A
dR6G
Green
BamHI
G^GATTC
G
dR110
Blue
XbaI
T^CTAGA
C
dTAMRA™
Yellow
XhoI
C^TCGAG
T
dROX
Red
HaeIII
GG^C
None
0
200
400
600
™
800
1000
1200
1400
3200
2800
2400
2000
1600
1200
800
400
0
Figure 2. GeneMapper analysis results of BAC fingerprinting data using the new
GeneScan™1200LIZ® Size Standard. DNA fragments from 40 to 1000 base pairs were
analyzed with GeneScan 1200 LIZ Size Standard.
Sizing Precision
0.3
0.25
Standard Deviation
6. Separation of fragments on a capillary
electrophoresis instrument
0.2
Green
Blue
Orange
Yellow
0.15
0.1
0.05
0
Increasing Fragment Size
Figure 3. Sizing precision Comparison per Fragment
Sizing precision of various labeled control wheat BAC fragments labeled using the SNaPshot ® Kit
along with GeneScan 1200 LIZ Size Standard.
Figure 2 & 3 Data courtesy of Drs. M.-C. Luo and Y. Ma, Department of Plant Sciences, University of California at Davis
For Research Use Only. Not for use in diagnostic procedures.
© Copyright, 2008 Applied Biosystems. All rights reserved.
Applied Biosystems, AB (Design), GeneMapper, LIZ, and SNaPshot are registered trademarks and GeneScan, POP-7, ROX, and TAMRA are trademarks of Applied Biosystems or
its subsidiaries in the US and/or certain other countries. TaqMan is a registered trademark of Roche Molecular Systems, Inc. Information subject to change without notice.
Printed in the USA. 12/2008 Publication 106AP25-01
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