Download AUTOMATED DNA SEQUENCING, MegaBACE 1000

Automated DNA sequencing
Summary
Screening and diagnosis of genetic diseases, development of new
therapies, and studying the effect of various drugs on our genes involve
screening for DNA mutations. Such Screening can be achieved by DNA
sequencing.
In the 1970s, Fred Sanger described the chain termination reaction or the
manual DNA sequencing system. Later other methods such as the
automated DNA sequencing were developed. These methods involved
fluorescence labeling technology as the slab –gel electrophoresis system.
Because the slab-gel systems were difficult to automate, capillary
electrophoresis systems were introduced, they saved time and yet were very
accurate; soon the system was enhanced by the multiple capillary array
technology.
The MegaBACE 1000 is a high throughput automated fluorescence gene
analysis instrument. It has a 96 capillary array system. in general the
instrument can be divided in to three compartments: the electrophoresis
compartment where separation of DNA fragments in the capillary arrays
takes place, the light collection compartment for sampling fluorescence
from each capillary and emission beamsplitter and filter compartment
that splits the emitted fluorescence and filters a specific range of light and
finally detects the light and converts it into electrical current which is
represented by colored traces in graphs called electropherogram.
Amersham Biosciences innovated DNA sequencing enzymology and
fluorescent dye chemistries to produce unique kits for MegaBACE 1000
With excellent properties; the sequencer has also user friendly software
for both instrument control and data analysis.
MegaBACE 1000 is highly automated, very sensitive and accurate with
flexible and easy software.
Paternity studied, forensic medicine investigations and genetic diversity
are also possible to be performed through microsatellite genotyping and
SNP analysis capabilities of the MegaBACE 1000.
Contents
1. INTRODUCTION……………………………………………………….1-4
1.1. STRUCTURE OF THE DNA MOLECULE……………………………………..1
1.2. DEFINITION AND FUNCTION OF THE DNA POLYMERASES……………2
1.3. POLYACRYLAMIDE GEL ELECTROPHORESIS……………………………3
1.4. THE BASES OF FLUORESCENCE TECHNOLOGY………………………….4
2. DNA SEQUENCING DEVELOPMENT……………………………….4-8
2.1. MANUAL DNA SEQUENCING………………………………………………...4-5
2.2. AUTOMATED DNA SEQUENCING…………………………………………...6-7
2.3. CAPILLRY SEQUENCING……………………………………………………..7-8
3. THE MegaBACE 1000 DNA SEQUENCER……………………………. 8
3.1. COMPONENTS OF THE MegaBACE 1000 SYSTEM…………………………8
 THE MegaBACE INSTRUMENT COMPONENTS………………………………9-12
 THE SOFTWAR PROGRAMS AND FUNCTION………………………………12-13
3.2. MegaBACE 1000 REAGENTS AND KITS…………………………………..13-15
3.3. THE INSTRUMENT’S OUTPUT………………………………………………...15
3.4. THE PRINCIPLE OF MegaBACE 1000 OPERATION……………………..16-17
3.5. PROPERTIES OF MegaBACE 1000 INSTRUMENT…………………………..17
4. APPLICATIONS OF THE MegaBACE 1000 SEQUENCERS……...17-18
5. REFERENCES………………………………………………………...19-20
1. INTRODUCTION
1.1. STRUCTURE OF THE DNA MOLECULE
The DNA molecule is made up of two poly nucleotides that form a double helix; each
polynucleotide consists of monomers called nucleotides. A nucleotide consists of a nitrogen
base, a pentose and a phosphate group. The portion of this unit with out the phosphate group is
called a nucleoside. The bases are of two families: pyrmidines and purines, members of the
pyrmidine family are the cytosine (C) and thyamin (T) but the purines are adenine (A) and
guanine (G).
The pentose connected to the nitrogenous base is a deoxy ribose sugar, because the atoms
in both the sugar and base are numbered, the sugar atom have a prime (′) after the number to
distinguish them, thus the second carbon in the sugar ring is 2′ and the carbon that sticks up from
the ring is called 5′ carbon. Nucleotides are linked together by covalent bonds called
phosphodiester linkage between the OH group on the 3′ carbon of one nucleotide and the
phosphate on the 5′ carbon of the next. The two free ends of the polymer are different from each
other. One end has a phosphate attached to the 5′ carbon, and the other end has a hydroxyl group
on the 3′ carbon. These are referred to as the 3′ end and 5′ end.
The two sugar phosphate backbones run in opposite directions from each other (5′→3′,
3′→5′) an arrangement referred to as antiparallel,the sugar phosphate backbone are to the outside
of the helix while the nitrogen bases are in the interior of the helix . The two poly nucleotides are
held together by hydrogen bonds between the paired bases, only certain bases in the double helix
are compatible with each other. Adenine always pairs with thyamin and guanine always pairs
with cytosine. (Figure 1) (Campbell et al, 2005)
http://nobelprize.org/medicine/educational/dna/b/replication/dna_structure.html
FIGURE 1: DNA STRUCTURE
1
1.2. DEFINITION AND FUNCTION OF THE
DNA POLYMERASES
Within each cell there are several types of DNA polymerases; DNA polymerase is an
enzyme that assists in DNA replication. Such enzymes catalyze the polymerization of
deoxyribonucleotides alongside a DNA strand, which they read and use as a template. The newly
polymerized molecule is complementary to the template strand and identical to the template's
partner strand. It only recognizes the regular sugar-phosphate portion.
fajerpc.magnet.fsu.edu/.../ image008.jpg
FIGURE 2: DNA POLYMERASE REACTION
All DNA polymerases synthesize DNA in the 5' to 3' direction. No known DNA
polymerase is able to begin a new chain. They can only add a nucleotide onto a preexisting 3'OH group. For this reason DNA polymerase needs a primer at which it can add the first
nucleotide. (Figure 2 & 3)
www.vetmed.iastate.edu/.../ polymerization.jpg
FIGURE 3: DNA SYNTHESIS
1.3. POLYACRYLAMIDE GEL ELECTROPHORESIS
Polyacrylamide gel electrophoresis is a class of zone electrophoresis where a solution is
applied as spot or band and the particles migrate through a solvent that is supported by inert and
homogenous medium as gels. Polyacrylamide gel is prepared by cross linking acrylamide with
N, N′ methylenebisacrylamide. There are two types of polyacrylamide gel electrophoresis,
column gels and slab gels. In slab gels the gel is allowed to harden in place and a comb is
suspended to top of the frame during hardening to form sample wells. After electrophoresis the
gel is stained and the slab is removed, the stain couples to the target molecule which is then
detected. (Figure 4)
web.siumed.edu/.../
images/chapter6/F0
6-21.jpg
FIGURE 4: SLAB GEL ELECTROPHORESIS
In column gels the gel is polymerized in a column and placed in a buffer reservoir. After
electrophoresis the gel is pushed out of the column and stained. (Freifelder, 1935). When a thin
layer of solution is placed in contact with a semisolid medium and an electric field is applied the
gel works as a molecular sieve and separates the molecules according to size. (Figure 5)
www.stanford
.edu/.../
gentest/f_s02g
elelect.gif
FIGURE 5: LOADING SAMPLES ON THE SLAB GEL
1.4. THE BASES OF FLUORESCENCE TECHNOLOGY
Fluorescence is the result of a process that occurs in certain molecules (generally
polyaromatic hydrocarbons or heterocycles) called fluorophores or fluorescent dyes.
A fluorescent probe is fluorophore designed to localize within a specific region of a biological
specimen or to respond to a specific stimulus. The fluorescence labels can be used in direct
labeling of nucleic acids by incorporating a modified nucleotide (2 deoxyuridin 5′ triphosphate)
containing an appropriate fluorophore.(Strauaan and Read,1999)
A photon of a specific energy is supplied by an external laser source that is absorbed by the
fluorophore creating an exited electronic state which exists only for about 1-10 nanoseconds.
When the fluorophore returns to its ground stat, a photon of lower energy and therefore longer
wave length than the excitation energy is emitted.
There are many different fluorophores each with different color; the emission wavelengths are
usually a little longer than excitation wavelengths. (http:// probs.invitrogen.com).
2. DNA SEQUENCING DEVELOPMENT
DNA sequencing was first developed in the 1970s by two groups of scientists. The
chemical cleavage reaction was developed by Allan Maxam and Walter Gilbert, and the chain
termination method was described by Fred Sanger the same biochemist who worked out Nterminal peptide sequencing in the 1950 s. (Miesfeld, 1999).
Genome sequencing depend on development of automated processes, Lee Hood and his
colleagues pioneered automated DNA sequencing using fluorescence detection system with a gel
electrophoresis system. This instrument was commercialized by Applied Biosystem Incorporated
(ABI) and it became very useful for the human genome project (Miesfeld, 1999).
2.1. MANUAL DNA SEQUENCING
The Sanger’s manual DNA sequencing method also known as the chain terminating
method depends on the synthesis of a new DNA strand using the DNA polymerase enzyme, this
enzyme is inhibited afterwards and synthesis of the chain is terminated (Sanger et al,1977) .
The DNA to be sequenced is provided in a single strand form. A primer binds specifically to the
3′ region of the desired DNA sequence. In the presence of the normal nucleotide precursors
(ATP, GTP, CTP, TTP) the complementary DNA strand is synthesized in 5′to 3′ direction. The
synthesis is carried out in the presence of base specific dideoxynucleotides (ddNTPs) which are
analogs of the normal dNTPs but differ in that they lack a hydroxyl group at the 3′ carbon
position as well as the 2′ position.
A dideoxynucleotide can be incorporated in to the growing DNA chain by forming a
phosphodiester bond between its 5′ carbon atom and the 3′ carbon of previously incorporated
nucleotide. Scince ddNTPs lack a 3′ hydroxyl group, any ddNTP that is incorporated into a
growing DNA chain can not participate in phosphodiester bonding at its 3′ carbon atom thereby
causing termination of chain synthesis. By performing four separated base- specific reactions
using a mix of all dNTPs and a small proportion of one of the four ddNPTs chain termination
will occur at one of the positions containing the base. Each one of the four base specific reactions
will generate a collection of labeled DNA fragments of different sizes with a common 5′end but
a variable 3′ end (the 5′end is defined by the sequencing primer and 3′end is variable because
insertion of a dideoxynucleotide occurs randomly) fragments that differ in size even by a single
nucleotide can be separated on polyacrylamide gel. The differently sized fragments can be
detected by incorporating labeled nucleotides, the sequence can then be read off by reading from
the bottom of the gel to the top, and this direction gives 5′→3′ sequence of the complementary
strand of the provided DNA template. (Figure 6) (Strachan and Read, 1999).
www.bioteach.ubc.ca/. ../sequencing2.gif
FIGURE 6: MANUAL DNA SEQUENCING
2.2. AUTOMATED DNA SEQUENCING
Modern versions of Sanger’s original technique generate fluorescently labeled DNA
fragments which are read in an automated fashion with electronic data capture. All possible
fragment sizes from 25 base pairs up to 1000 bases are produced by the sequencing chemistry
with each fragment containing a single fluorescent label that corresponds to the last DNA letter
of the fragment and by matching the color to size the original DNA sequence can be easily
determined. (Hawkins et al, 2000)
The automated procedure generally uses primers or dideoxynucleotides to which fluorophores
are attached. Four separate fluorescent dyes are used as labels for the base specific reactions; this
permits the sequencing reaction to be preformed in a single tube and the product to be resolved
in one lane. During the electrophoresis run a laser beam is focused at a specific constant position
on the gel. As the individual DNA fragments migrate passed this position the laser causes the
dye to fluoresce. Maximum fluorescent occurs at different wavelengths for the four dyes and the
information is recorded electronically and the interpreted sequence is stored in a computer
database. (Strachan and Read, 1999)
Among the first automated DNA sequencers were the slab gel electrophoresis instrument that
utilized a thin cross linked polyacrylamide gel sandwiched between two glass plates, fluorescent
labeled fragments were manually loaded to the top of vertical gel and electric filed was applied,
as the negatively charged DNA fragments migrated through the gel they were sized and
fractionated by the polyacrylamide gel. The fragments were automatically excited with a
scanning argon laser and detected by a camera.
www.biochem.arizona.edu/.../
media/fig.9.1-2.jpg
FIGURE 7: AUTOMATED DNA SEQUENCING
FIGURE 7: AUTOMATED DNA SEQUENCING
2.3. CAPILLARY SEQUENCING
http://www.unileipzig.de/~strotm/equipment/megabace/MegaManual.pdf
FIGURE 8: A CLOSER LOOK AT THE
CAPILLARIES
Because the slab gel electrophoresis was still manual and needed several steps,
modern capillary systems were introduced. These systems are currently the fastest system
available they use the same basic electrophoresis and detection method technology as the slab
gels with several added advantages, each sample is separated and analyzed in an individual
capillary, this eliminates sample tracking issues, gel uniformity problems, heat dissipation from a
capillary is much more efficient due to its higher surface –area-volume ratio and this allows
higher run voltages to separate DNA fragment faster than slab gels.(Hawkins etal,2000).
Capillary gel electrophoresis (CGE) can be basically regarded as separation of molecules in a
single lane. In CGE fused silica capillaries of 50-100 µm in diameter and 30-80 cm in length are
filled with a separation matrix consisting of a gel and electrode buffer. Two types of gels are
used: chemical gels in which the pore structure is dynamic and formed by covalent binding
(eg.polyacrylamide) and physical gel entanglement of polymers (as liner polyacrylamide).
(Figure 8).
Solution phase DNA molecule are injected into the capillary either by pressure or electrokienetic
injection and separated inside the capillary according to their size under very high voltage
conditions (5-30 kv). The use of such voltage is possible due to rapid heat dissipation of the
capillary and hence electrophoresis time is very short. The molecules are typically detected using
UVlight absorption or laser induced fluorescent detection as they pass a detector at the end of the
capillary. The sample data are collected and stored by a computer and usually analyzed using
dedicated software. (Larsen etal, 2000).The throughput of CE may be enhanced by using
multiple capillaries arranged in parallel arrays. Among the 96 capillary array instruments are the
ABI 3700 Applied Biosystem and MegaBACE 1000 Amersham Pharmacia Biotech. (Larsen
etal, 2000)
3. THE Mega BACE 1000 SEQUENCING SYSTEM
The MegaBACE 1000 is a high throughput automated gene analysis instrument with
a 96 capillary array system based on four color imaging fluorescence detection through a
scanning confocal imaging system.
3.1. COMPONENTS OF THE MegaBACE 1000 SYSTEM
The MegaBACE 1000 system consists of:
 The hardware component, which includes the MegaBACE instrument, the power supply
and fans, the computer, monitor key board and mouse, nitrogen pressure source. (Figure
9).
 The software component.
http:// www 4.Amersham biosciences.com
FIGURE 9: THE MegaBACE SYSTEM AND COMPONENTS
THE MegaBACE INSTRUMENT COMPONENTS
 The electrophoresis compartment:
It consists of a cathode array stand where an array of 16 capillaries assembles into a
cathode bar; each capillary is then contacted to the surface of the sample plate. (http:// www
4.Amersham biosciences.com). The instrument utilizes 6 arrays of 16 capillaries that provide
rapid parallel separation of the dye labeled DNA fragments on a total of 96 samples. The
capillaries are filled with a replaceable non cross-linked linear polyacrylamide (LPA) matrix.
The instrument applies a voltage pulse to electrokinetically inject all 96 samples from a
microtitre plate simultaneously. (Figure 10)
(http://www.uni-leipzig.de/strom/equipment/megabace/Mega Manual.pdf)
http:// www 4.Amersham biosciences.com
FIGURE 10: A CAPILLARY ARRAY
The electrophoresis compartment also contains a capillary detection window; each capillary has
a clear detection window located at a fixed distance from the sample loading point through
which the samples are scanned. Finally, it contains the anode reservoir holder that contains an
anode cover and a plug, and holds six matrix tubes. (Figure 11) ().
www.jgi.doe.gov/education/ how/electrophoresis.gif
FIGURE 11: THE ELECTROPHORESIS COMPARTMENT
 Laser, scanner and light collection system:
During electrophoresis the instrument uses laser excitation and a potent confocal
optical detection system to both excite and detect the labeled DNA fragments as they migrate
passed the detection window. Confocal laser scanning focuses on each sequencing product
within the capillary, this enables the production of data beyond 800 bases (in three hours run)
with very sensitive detection capabilities.
(http://www.uni-leipzig.de/strom/equipment/megabace/Mega Manual.pdf).
The excitation light is focused on the sample by an objective lens, and the emitted light is
collected by the same lens.
The detection windows of the capillaries lie in the focal plane of the objective lens
which focuses the laser light to a point in the focal plane called the focal point. In an array of
capillaries only the capillary at the focal point is illuminated, and along the length of the
capillary only the part of the sample at the focal point is illuminated .The fluorescent light from
the sample passes back through the microscope objective lens .The objective lens and an
additional lens within the instrument focus the light from the focal point to a second point called
the confocal point. The small hole at the confocal point allows light from the focal point to pass
through the photomultiplier. Light emitted out side the focal point is not in focus at the confocal
point and therefore rejected. (Figure 12) (http:// www 4.Amersham biosciences.com)
The excitation options include:
 Blue laser mode: excites up to four dyes at 488 nm.
 Green laser mode (dual laser instrument): excites up to four dyes at 532 nm.
 Green and blue laser mode (dual laser instrument): uses the blue laser to excite two dyes at
488 nm and the green one to excite the other two dyes at 532nm. (http://www.unileipzig.de/strom/equipment/megabace/Mega Manual.pdf)
http:// www 4.Amersham biosciences.com
http://www.unileipzig.de/~strotm/equipment/mega
bace/MegaManual.pdf
FIGURE 12: THE LIGHT COLLECTION
SYTEM
 Emission beamsplitter, filters and photomultiplier:
To record four dyes separately, the MegaBACE instrument uses two optical sets each
consisting of a beamsplitter plus two filters. The beamsplitter separate light by wave length
cutoff. Light with wavelengths longer than the cutoff passes through the beam splitter and light
with wavelengths shorter than the cutoff is reflected by the beam splitter.
Because the beamsplitter separation is imperfect and the overlapping of emission from several
dyes; the instrument uses emission filters. There are two types of emission filters:
 Band pass/ it rejects most of the light with wavelength shorter than a specified cutoff and
longer than a second specified cutoff this allows light of wavelengths between the two
cutoffs to pass through.
 Long pass/ rejects light with wavelengths shorter than specified cutoff and allows light of
longer wavelengths to pass through (http:// www 4.Amersham biosciences.com)
Photomultiplier (PMT)/ the instrument use the photomultiplier tubes to collect the filtered light
the PMT converts light energy into electrical current. The MegaBACE instrument uses four
spectral channels to detect the emission of the multiple dyes in each capillary. A
spectral channel is the combination of beamsplitter, emission filters and PMT. (Figure 13)
http:// www
4.Amersham
biosciences.co
m
http:// www
4.Amersham
biosciences.com
FIGURE 13: BEAMSPLITTERS AND FILTERS
THE SOFTWARE PROGRAMS AND FUNCTION
The MegaBACE 1000 DNA sequencing system has user friendly software for
instrument control and data analysis, the instrument control manager software and the sequence
analyzer software have been designed for DNA sequencing.
Instrument Control Manager Software (ICM):
It has a flexible graphical user interface that provides total control of the system. It
controls all the run parameters as sample injection, electrophorisis, temperature, gel matrix
replacement, and optic configuration.
It also edits the run parameters and sample sheet information, saves multiple run conditions,
accesses the saved runs by using a pull down menu and displays real-time run data for quick and
easy instrument monitoring.
The ICM provides a smooth workflow that is divided into three windows; the plate setup
window for entering run parameter, the instrument control window for running protocols and the
run image window for viewing data collection.
The Sequence Analyzer Software:
It gives the power to handle genomic data, it can analyze data or export data to other files for
analysis, and it can retrieve data from as many as 20 runs and analyze up to 1920 samples at a
time. It also can evaluate the quality of base calling, which is preformed using UTAH base caller
component from Cimarron software Inc. After base calling sequence analyzer calculates a
quality index for entering sequence that is displayed above each base as a curve within the
electropherogram.
(http://www.uni-leipzig.de/strom/equipment/megabace/Mega Manual.pdf)
The sequence analyzer shows all operations in one screen with windows for plate folder
information, individual trace information and trace viewing. The output from several different
base callers can be displayed; up to 10 different base callers can be installed. Cimarron 3.12 base
callers has been improved in many ways , it is more accurate and base calls at lower signals,
reads short PCR fragments, and increased spectral separation.
3.2. MegaBACE 1000 REAGENTS AND KITS.
Separation matrix: MegaBACE long read matrix with linear polyacrylamide (LPA) is
optimized for both sequencing and genotyping, it saves time when switching between
applications, and results in less waste. It can produce DNA sequence of 700 bases or resolve
multiplexed fragments in excess of 900 bases. The gel matrix is replaceable and each pack
includes
96
pre-dispensed
tubes
plus
running
leipzig.de/strom/equipment/megabace/Mega Manual.pdf)
buffer.
(http://www.uni-
Sequencing reagents: Amersham Biosciences innovated DNA sequencing enzymology and
fluorescent dye chemistry. These innovations are brought together in DYEnamic ET terminator
and dye primer kits. To run a sequence with this kit, a reaction premix is combined with a
laboratory’s template DNA and primer, the single reaction mixture is thermally cycled. After
cycling the reaction products are precipitated with salt and ethanol or they are passed through a
gel-filtration resin and concentrated to remove the unincorporated dye labeled terminators. The
reaction products are resuspended in a formamide loading buffer and then separated and detected
in the MegaBACE 1000.
The kit’s components include:
1) DYEnamic ET terminators
Each dideoxy terminator is labeled with two dyes. One of these dyes fluorescein, has
a large extinction coefficient at the wave length 488 nm of the argon ion laser. In the instrument
the fluorescein donor dye absorbs light energy and transfers the collected energy to an acceptor
dye. Each of the four chain terminators ddG, ddA, ddTand ddC has a different acceptor dye
coupled with the fluorescein donor. The acceptor dye then emits light at their characteristic wave
lengths. The fluorescence is detected by the instrument which allows the nucleotides that caused
the termination to be identified. Using the energy transfer provides a more efficient excitation of
the
acceptor
dyes
than
dose
using
direct
excitation
by
laser.
(http://www.uni-
leipzig.de/strom/equipment/megabace/Mega Manual.pdf)
2) Thermosequensase DNA polymerase.
This enzyme is specifically engineered for DNA sequencing and it has many properties which
include:
- Tolerance to high salt conditions.
- Efficient utilization of dITP.
- High processivity.
- Excellent performance on GC rich templates.
The enzyme formulation contains thermostable inorganic pyrophosphatase (TAP) cloned from
the thermophile thermoplasma acidophilum. TAP hydrolysis the inorganic pyrophosphate
product of nucleotide polymerization which prevents pyrophosphorolysis, the reversal of
polymerization from occurring, pyrophosphorolysis can result in sequence data with missing
peaks.
3) DYEnamic ET primer reagents.
These reagents should be combined with the template DNA and dye labeled primers before
detecting. The energy transfer dye primers are oligonucleotide DNA sequencing primers that
have two fluorescent dyes attached for improved detection properties, one dye called the donor
dye was chosen because it efficiently absorbs light of the wave length of the argon ion laser. This
dye transfers absorbed light energy to the second dye attached to the primer. The acceptor dye
emits the absorbed energy as fluorescence at its normal emission wavelength. Because all the
primers absorb the laser light efficiently the effective fluorescence of the ET primers are 2 to 12
times
greater
than
the
primers
having
single
dye
labels.
(http://www.uni-
leipzig.de/strom/equipment/megabace/Mega Manual.pdf)
3.3. THE INSTRUMENT’S OUTPUT
The output from the spectral channel is represented by colored traces in graphs called
electropherograms. Each electropherogram represents data from a single capillary (sample) and
consists of four colored traces, one for each spectral channel. The bases correspond to display
colors for each type of dye in sequence analyzer. The same color scheme is used to display the
raw and base called traces. The color of each base is constant. A= GREEN, C= BLUE, T= RED,
G= BLACK. (Figure 14)
users.rcn.com/.../ D/DNA_sequence.gif
www.bioteach.ubc.ca/.../ automatedseq.gif
FIGURE 14: ELECTROPHEROGRAMS
3.4. THE PRINCIPLE OF MegaBACE 1000 OPERATION
The Mega BACE 1000 is based on four color fluorescence detection through a
scanning confocal system. Capillaries have a 75µm inner diameter and are 40 cm from injection
to detection region. A 96 sample capacity is obtained with six replaceable arrays each consisting
of 16 capillaries. Each capillary runs directly from the sample plate well through a temperature
controlled chamber in to the confocal optical scanner where it joins the other 95 capillaries in
forming a linear array. In this region the capillary coating has been removed to allow the
confocal scanner optical access to the interior of the capillaries. Down stream of the scanning
region, each set of 16 capillaries is bundled together and terminates in tubes of separation matrix
buffer. During operation a confocal objective scans back and forth over the 96 capillary arrays,
thus sampling fluorescence from each capillary by directing a laser beam of 488 nm /532nm into
the center of the capillary through the capillary wall and collecting any resulting fluorescence the
fluorescence is color separated and collected using photomultipliers. The separation matrix
consists of liner polyacrylamide gel (LPA) provided by Amersham. LPA has excellent separation
properties and it is injected into the capillaries from the anode and under pressure from a
compressed (1000Ib/In2) nitrogen gas cylinder. Samples are loaded into the instrument in a single
96-well-plate that is manually placed in a drawer at the bottom of the instrument. Once the
drawer is closed, it is lifted (with gas from a second cylinder at 120Ib/In2 and brought against the
end of the 96capillaries and 96 cathode electrodes. Loading of the matrix tube is similar. The
DNA samples are electrokinetically injected by applying 3-6 Kv for 10-60s. Because samples are
injected directly from sample plate into the capillary, each individual capillary and electrode
must contact the surface of the sample this requires each well of the sample plate to contain at
least 5μl of fluid. (Marziali and Akeson, 2001). (Figure 14).The instrument uses two beam
splitters to split the emitted fluorescence light and then filters the light through four filters. Each
filter permits only a specific range of light, corresponding to the emission of one dye to pass
through the PMT tube. Two PMTs detect the filtered light and convert the light into an electrical
current which is digitalized to produce an electropherogram for each capillary. (http:// www
4.Amersham biosciences.com)
http://www.uni-leipzig.de/~strotm/equipment/megabace/MegaManual.pdf
FIGURE 14: INSIDE MegaBACE
3.5. PROPERTIES OF MegaBACE 1000 INSTRUMENT.
 Capillary electrophoresis with automated gel matrix replacement, sample injection, DNA
separation and base calling. There is no risks of gel pouring, no need for washing glass plate or
retracting samples.
 Electrokinetic sample injection automatically loads sample from the 96 well-plate into
capillaries, this eliminates sample mix-up.
 The energy transfer dye chemistry kits are very sensitive and accurate.
 LPA is innovation in capillary electrophoresis sieving matrices that enables read lengths in
excess of 800 bases.
 It has flexible, user friendly analysis software that produces accurate base calling data.
1. APPLICATIONS
SEQUENCERS
A.
OF
THE
MegaBACE
1000
MICROSATELLITE GENOTYPING
Microsatellites are di, tri or tetra nucleotide tandem repeats in DNA sequences. The
number of repeats is variable in populations of DNA and within alleles of an individual, if a
microsatellite is flanked with fluorescent, PCR amplification will give a pair of fluorescent
allelic product which will vary in size according to their repeat length. Microsatellites are used as
genetic markers that can be used for genetic diversity, identifying important genetic trait,
forensic and paternity studies.
The MegaBACE genotyping system includes the genotyping test kit which provides standardized
reagents for spectral separation, also the MegaBACE genetic profiler score card software that
scores the quality of result from the test plate and evaluate key areas of instrument performance.
It can also summarize information from plats and verify the over all performance of the system.
(http:// www 4.Amersham biosciences.com)
B.
SNP ANALYSIS
Single nucleotide polymorphism or SNPs are DNA sequence when a single nucleotide
sequence in the genome is altered. SNPs make up about 90% of all variations; SNP occurs every
100 to 300 bases along the human genome. SNPs occur in both coding and non-coding sequence,
many SNPs have no effect in cellular functions but so many others can predispose people to
disease or influence their response to drugs.
SNP maps may help scientist to identify the multiple genes associated with cancer, diabetes,
vascular and mental diseases.
The MegaBACE SNP genotyping kit enables four color SNP genotyping of PCR products with
the DNA analysis system the kit contains thermosequnase DNA polymerase and four dye labeled
ddNTPs in a single tube.
The SNP analysis system can load twelve 96-well sample plates over a period of about 30
minutes. A multiple injection marker is added to each sample prior to injection .A total of 1200
SNP genotypes can be obtained in less than two hours. MegaBACE SNP profiler V 1.0
window based software provides automatic allele calling, editing and verification function,
processing
signal
data
and
outputting
SNP
genotype.
(http://
www
4.Amersham
biosciences.com)
5. REFERENCES
 Campbell, A.Reece, B. (2005). Biology. United States of America. Pearsons Education,
seventh edition. p88
 David Freifelder. (1935). Physical biochemistry and molecular biology. United States of
America. WH freeman and company. Second edition.p 282 – 283
 Delbruck, M (1945) on the replication of Deoxyribonucleic Acid (DNA) Proc.Natl.Acad.Sci
40: 783 – 788
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