中南大学本科论文
中文摘要
中南大学
本科生毕业论文
题
目
浸矿体系微生物群落基因组芯片的构建与评
估
学生姓名
指导教师
学
院
谢
明
刘学端 教授
资源加工与生物工程学院
专业班级
生物工程 0202
完成时间
2006 年 6 月 22 日
1
Index
Abstract in Chinese ··········································································· 1
Abstract in English ··········································································· 2
Chapter 1 Introduction ······································································· 3
Chapter 2 Review ············································································· 4
2.1 Challenges in microbial community analysis ·································· 4
2.2 Molecular methods in microbial community research: development and
limitations ······················································································ 5
2.2.1 Conventional molecular methods in microbial analysis ················· 5
2.2.2 Microarrays for research in microbial community ······················· 6
2.3 Conclusion and future trend toward microbial community study ··········· 8
Chapter 3 Materials and experiment ······················································· 9
3.1 Materials and instruments used in experiments ································ 9
3.2 Bacteria cultivation and the extraction of genomic DNA ···················· 10
3.3 CGA fabrication ···································································· 12
3.3.1 Sample Pretreatment: target DNA preparation ··························· 12
3.3.2 Microarray fabrication ······················································· 12
3.4 Hybridization of CGA for examining its sensitivity, specificity and
quatitative potential ··········································································· 13
3.4.1 Fluorescent labeling of Probe ··············································· 13
3.4.2 Hybridization of labeled probe to CGA ··································· 14
3.4.3 Scanning of hybridization result ············································ 15
Chapter 4 Results and discussion ··························································· 15
4.1 The quantitation of probe DNA concentration ································· 15
4.2 Surface scanning of CGA after hybridization ·································· 16
4.3 CGA capability evaluation ························································ 18
4.3.1 Evaluation of CGA sensitivity ·············································· 18
4.3.2 Evaluation of CGA specificity ·············································· 18
4.3.3 Evaluation of CGA quantitative potential ································· 19
Chapter 5 Conclusion ········································································ 23
Acknowledgement ············································································ 25
References ····················································································· 25
摘
要
用传统分离培养方法进行浸矿体系微生物群落结构分析工作量大，耗时多。
而常规分子生物学方法虽然灵敏度、精确度高但平行测试能力有限。基因芯片技
术为复杂样品分析提供了强大的工具, 而基于基因芯片技术发展的群落基因组
芯片为检测自然环境微生物群落种群数量及其相对丰度提供了新的思路和方法。
本论文提取来自细菌Leptospirillum属的10个菌株，Acidithiobacillous Ferrooxidan
种的9个菌株，以及Acidiphilum属的5个菌株的基因组DNA构建了浸矿微生物群落
基因组芯片。优化了芯片检测的杂交反应条件。对所构建的芯片的灵敏度、特异
性以及定量性能进行了评估。结果显示所构建的群落基因组芯片特异性良好,几
乎不存在交叉杂交；芯片灵敏度达到0.11ng左右，高于文献报道水平；杂交信号
强度与探针样品浓度之间存在较好的线性关系，可以用于定量分析。
关键词：基因芯片；群落基因组芯片；微生物群落；生物浸出
中南大学本科论文
英文摘要
Abstract
The identification for the community structure in bioleaching systems used to be a
fatiguing and time-consuming task. The newly developed method—Community
Genome Array (CGA), which is based on microarray technology, provides a
brand-new strategy for the detection of microorganism populations, their presence and
abundance in bioleaching systems. In this assay, Community Genome Array
containing genomic DNA from 10 strains of Leptospirillum, 9 strains of
Acidithiobacillous Ferrooxidan, and 5 strains of Acidiphilum is designed and
constructed. The reaction condition for the hybridization of this CGA is carefully
examined and optimized. The capability of this Community Genome Array is tested in
terms of sensitivity, specificity and quantitative potential. Results suggest that the
CGA fabricated is highly sensitive as it can detect 0.11ng of sample DNA; little cross
hybridization is observed, suggesting that the CGA is capable of differentiating DNA
of bacteria in the species level. Besides, linear relationship between the hybridization
signal and probe DNA concentration is obtained, thus, the CGA fabricated can be
applied in quantitative analysis.
Keywords: mcrioarray; community genome array; microbial community; bioleaching
system
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中南大学本科论文
第 1 章 前言
Chapter 1 Introduction
In recent decades, microorganisms in bioleaching systems have aroused more and
more interest in researchers because of their potential in alleviating environmental
pollution as well as extracting and refining minerals (such as Fe, Cu) from low grade
ores. Generally, microorganisms in bioleaching systems are chemolithoautotrophic,
Gram-negative, aerobic bacteria (there are existence of aechaea and even eukaryotes
though) grow in extremely acidic fluids (typical pH values are within 1.8~3.5)
containing abundant minerals. This kind of ecology system is proved to be simpler
than common fields because it owns fewer kinds of substances, and there are very
little organic compounds. Microorganisms in bioleaching systems obtain the energy
required for their metabolism and reproduction mainly through the oxidation of
inorganic substance such as Fe2+, sulfur and the reduction of Fe3+, etc. Dominant
bacteria in bioleaching systems are typically: Acidithiobacillous Ferrooxidan (At.f),
Leptospirillum (L), and Acidiphilum (A.p). There are also archaea in such
environments, but the bulk of these archaea belong to genus Thermoplasmatales and
Sulfolobales. Besides, There have been a few reports of the presence of eukaryotes [1].
In this assay, we focus on the detection and analysis of bacteria in the community
level: their presence and relative abundance, and the interaction of organisms within
the community and with the environment.
The distinct environment in which these microorganisms live and reproduce has
tremendously perturbed the course of detecting, identifying certain microorganisms
and analyzing the structure & metabolism networks for the community of interest.
Conventional methods, which are originally designed for research of microorganisms
grown in modest environment, cannot be applied to these systems because of the
harsh environmental condition (extremely acidic fluid, the lack of organic substance,
high temperature, etc). For example, a newly developed method—Biolog System
(first designed by Biolog Company), which is capable of identifying pure culture and
analyzing different microbial community with high accuracy [2], proved to be limited
for the detection and analysis of bacteria in bioleaching systems because these
microorganisms do not utilize carbon as their main source of energy. For the reasons
above, microarray becomes a ideal research tool for the analysis of microbial
community in bioleaching systems because it has no requirements for the metabolism
style of organisms to be tested, which means this method is relatively unbiased and
non-directed. This novel type of technology has offered a brand-new method which is
capable of detecting hundreds, even thousands kinds of microorganisms at one time
(high throughput) with high sensitivity, specificity and accuracy. Besides, with the
help of computer, we will be able to quantify the information obtained from the
hybridization of microarrays, as there have been several kinds of microarrays that
already proved to be potentially quantitative, such as Functional Gene Array (FGA)
and Community Genome Array (CGA).
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中南大学本科论文
第 2 章 综述
Our goal here is to construct a Community Genome Array in order to identify the
community structure of the sample obtained directly from bioleaching systems.
Compared to traditional method in depicting the community structure in natural
environment, which may take years to isolate bacteria within the system, analysis
based on CGA will cost only several days to comprehensively reveal the community
structure information. However, the microarray we fabricated here is a prototype
CGA, as it contains only 24 strains of bacteria; but in later experiments, genomic
DNA from other bacteria will be successively planted on other chips and thus the
capability of the CGA will be enhanced. Although Community Genome Array is
powerful in detecting and identifying microorganisms, the design and construction of
this type of microarray require special technology and equipment. Moreover, the
hybridization condition needs to be carefully examined because some subtle change in
hybridization condition (temperature, solution, etc) may produce data that are
remarkably different, which may lead to inaccurate conclusion of the community
under investigation. After fabricating this CGA, we evaluated its capacity, sensitivity
and quantitative potential. Suggestions are made for promoting CGA performance.
Chapter 2 Review: the development of methods in microbial
community analysis in bioleaching systems
2.1 Challenges in microbial community analysis
The study of microbial community can offer us the overall information about the
microorganisms of interest, their metabolic function, genetics, species diversification,
evolution and regulatory networks. However, the traditional ‘isolate and study’
research strategy proved to be quite limited for analysis in community level, because
this strategy follows the way of simplifying a complex system while our effort is
aimed directly at the integrated system—the microbial community as a whole. Thus,
conventional research methods, which are centered with acquiring and maintaining
the pure culture of certain microorganisms, proved to be limited in the study of
microorganisms in the community level, especially for microbial community in
special environments (such as the AMD system and bioleaching systems). Besides,
other challenges occur as we are to explore microorganism and to obtain information
about the metabolic, genetic function and regulatory networks in their community
context. First of all, it is extremely difficult for us to acquire community structure
information that exactly represents the original sample because our ability to conduct
research in-situ is limited. This is of great importance for research works oriented at
environmental application (such as study concerning bioleaching systems and the
mitigation of environmental pollution), as the results are obtained from samples that
are not representative of microbial community in nature. For example, researchers
conducting experiments towards the AMD system have once reached the conclusion
that bacterium Acidithiobacillus Ferrooxidans is the dominant species in most AMD
systems. But recent report pointed out that the conclusion above may just brought by
the fact that bacteria of the Acidithiobacillous Ferrooxidan species is easier to
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中南大学本科论文
第 2 章 综述
cultivate and thus a significant part of experiments are designed for the detection and
analysis of these bacteria [3]. FISH experiment examining the microbial community
directly in the Richmond Mine, California, USA, revealed that the bacteria of species
Acidithiobacillus Ferrooxidan only takes up a small portion in the community, some
of the species which are thought to be of great amount in nature proved to be too few
to be detected [4]. Secondly, the preservation of genomic information for species that
are scarce in the community requires special technology. Though Polymerase Chain
Reaction (PCR) technology has greatly promoted analysis in molecular level, it is still
difficult for us to design proper primers to avoid biased duplication, which may lead
to false conclusion as the DNA of some minor populations in the community may
thrive because the primers are homologous and the real dominant species is neglected.
Thirdly, because of the diversity and the heterogeneity of microorganisms, limitations
arise due to the lack of reproducibility of experiments, which means the results gained
from former experiments may not match successive studies of exactly the identical
system. For all the reasons above, novel research strategy of unbiased analysis is
developed and methods with higher throughput, specificity, sensitivity and accuracy
are designed.
2.2 Molecular methods in microbial community research: development
and limitations
2.2.1 Conventional molecular methods in microbial analysis
An overall understanding of microbial community structure, the partitioning of
metabolic function among populations, and the roles each of the species performs in
the consortia will greatly enhance our ability to discover important metabolic function
and the regulation of microbial activity. Genomic study and molecular methods will
provide insights into the essence of the ecology and evolution of microorganism in
natural environment. We might be able to design novel cultivation strategy, which
may lead to successfully isolation of special microbial species. For example, utilizing
genomic information of the AMD system in Richmond Mine at Iron Mountain,
California, USA, researchers successfully isolated the microorganism Leptospirillum
group III [5]. In recent years, researchers have developed various kinds of methods
based on the metabolic and molecular character of microorganisms for the study of
microbial community. For instance, based on the different extent to which
microorganisms utilizing 95 kinds of carbon sources (metabolic level), the Biolog
system can build up the metabolic matrix depiction of certain microorganism and the
metabolic fingerprint of environmental sample. Besides, the development in
molecular methods has largely enhanced our ability to survey environmental
microbial community. Among these methods are Single-Strand Conformation
Polymorphism (SSCP), Denatured/Temperature Gradient Gel Electrophoresis
(DGGE/TGGE), Fluorescent In-Situ Hybridization (FISH), Terminal Restriction
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中南大学本科论文
第 2 章 综述
Fragment Length Polymorphism (T-RFLP), Reverse Sample Genome Probing
(RSGP), etc. Molecular methods are advantageous as they are generally more
sensitive, specific and accurate. The information obtained from these methods
provides insight into the genetic character of microorganisms in microbial community.
Besides, PCR technology has allowed experiment toward little DNA sample.
However, due to the diversity of microorganisms in natural environment (as we know
that 1 gram of soil sample may contain more than 1000 microorganism species), the
methods above seem to be weak in the detection and identification of all the species in
the community simultaneously, that is, their parallel capacity is limited. For example,
the newly ameliorated FISH: Multicolor FISH can detect only 7 kinds of target
genomic DNA fragments at one time. Further, as PCR manipulation is essential to
almost all the methods based on the hybridization of nucleic acid molecule (because
we cannot obtain enough amount of DNA to conduct these experiments directly from
cultivation), the biased PCR amplification, which we have no ideal solutions so far,
should be taken great consideration, as it may bring in feint information about the
community structure. Moreover, methods such a FISH, T-RFLP, SSCP, etc. require
stringent reaction condition, which resulted in the lack of reproducibility. Subtle
change in reaction temperature, pH value or solution constitution may produce data
with significant difference. In a word, all these methods owns its peculiar advantage
in certain aspect, but may be weak in other fields, thus, the combination of several
methods or the development of comprehensive new strategy may be promising for
research work in the future.
2.2.2 Microarrays: powerful tool for research in microbial community
Microarray technology-the production of the combination of chemistry, physics,
mathematics and bioinformatics, first designed by Mark Schena etc. in 1998,
developed rapidly in recent decade because of its great potential in analyzing samples
containing hundreds, even thousands kinds of DNA (or protein, etc.) at one time
(which is referred to as ‘high-throughput’) as well as detecting & identifying samples
with high sensitivity, specificity and accuracy. Generally, microarrays are chemically
modified smooth optic glass slides containing huge amount of special information
(such as the functional gene of bacteria, antibody of certain antigen, etc.). DNA
molecules are planted on the chip through electronic interaction with positive residue
or covalent binding on the surface of the slide. This novel technology can be utilized
to monitor gene expression, detect specific mutations in DNA sequences and
characterize microorganisms in environmental samples. The application of this new
technology will help us to penetrate the microbial community in various kinds of
natural environment. In recent years, professor Jizhong Zhou and his co-workers in
his laboratory in Oak Ridge National Laboratory, USA have designed several novel
kinds of microarrays which are referred to as Functional Gene Array (FGA),
Phylogenetic Oligonucleotide Array (POA), Whole-genome Open Reading Frame
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中南大学本科论文
第 2 章 综述
Arrays, and the Community Genome Array, etc. The comparison of the characteristics
of these four kinds of microarrays is listed in Table 2.1[6]. The Functional Gene Arrays
(FGAs) contain functional gene sequence information and are primarily used for the
functional analysis of microbial community activity in the environment. FGAs based
on PCR product is capable of differentiate genes possessing <80–85% sequence
identity, while FGAs based on oligonucleotides (about 50 base pair in length) can
clearly differentiate genes with less than 86–90% sequence identity using
hybridization conditions of 50 º C and 50% formamide [6]. The Phylogenetic
Oligonucleotide Array can provide phylogenetic information about the community
under investigation; and the Whole-genome open reading frame array can be used to
reveal genome diversity and relatedness among closely related organisms. The
Community Genome Array is distinctive among these various kinds of microarrays,
because it is fabricated using genomic DNA of the bacteria, which may more than
20kb in fragment length. CGA is specific enough to tell microorganisms in species,
even strain level under stringent condition [7](see reference 7 for detail). Besides, a
significant characteristic of CGA is that detection works using this sort of microarray
dose not require the sequence information of the probes fabricated on the chip
beforehand. This is of great importance and utility, because the lack of sequence
information of isolated microorganisms (the fact is little or nothing is known about
the majority of such organism) will no longer perturb research works of detecting the
presence and relative abundance of these microorganisms. For CGA analysis, once
some new kind of bacteria is isolated and identified, we can use its genomic DNA to
detect its presence in environmental sample without knowledge of its DNA sequence,
which will greatly reduce research workload. Considering DNA consumption, CGA is
also advantageous than traditional DNA-DNA reassociation approach, as its
fabrication needs only 2µg of DNA, while the latter method requires about 100µg of
DNA [6]. Despite all of the advantages of CGA, the capability of this sort of
microarray still depends on the isolation we obtained beforehand, because in order to
detect certain kinds of microorganisms, we must first acquire the pure culture in order
to extract genomic DNA we needed to fabricate the chip. Thus, more isolation we get,
more representative information about the community under concern we may obtain
through later on experiments. This reveals one important weakness in CGA: it is still
pure-culture dependent (which means it cannot be used for the detection of
uncultivated microorganisms) although the successive hybridization and detection
work can be carried out without culturing. Besides, there are other problems
concerning microarray analysis in microbial community, including CGA. ① :
Fluctuation in internal hybridization of microarray is critical, as it precludes
comparison of data between experiments conducted in different laboratory, even the
serial experiments in the same laboratory using the same strategy. ②:The threshold
of detection sensitivity is still not clear. ③: Data produced through microarray
hybridization may be extremely huge, but the development of tools for the analysis of
such data has fallen behind, thus it is difficult to reveal effective information from
data obtained; internal hybridization and background signal may bring false
conclusion [8].
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中南大学本科论文
第 2 章 综述
Table 2.1 Major differences of various types of microarrays for environmental studies
(contents of this table are excerpted from reference 6)
Probe size
Information
provided
Targeted
microorganisms
Specificity
Sensitivity (ng of
DNA)
Quantitation
Taxonomic
resolution
CGAs
PCR-based FGAs
Oligonucleiotide-based
FGAs
Whole Genomic
DNA
200-1000bp
50-70bp
Phylogenetic
Functional
Functional
Culturable and
non-culturable
<80%-85% sequence
homology
Culturable and
non-culturable
<86%-90% sequence
homology
About 0.2
About 1
About 8
Yes
Yes
Yes
Genus-species
Genus-species
Species-strains
culturable
species
2.3 Conclusion and future trend toward microbial community study
Although there has been great challenges in research of microbial communities in
environment, numerous new methods has provided powerful tool to penetrate the true
character of the metabolism, genetics, ecology, and regulatory networks of
microorganisms of interest, and to utilize these organism for environmental
application. Methods such as FISH have allowed in-situ research, which is critical in
revealing microbial community characteristic in natural environment [9]. However, our
ability to conduct in-situ study is still limited because of the complexity of natural
sample. Microarray technology has great potential as a specific, sensitive, quantitative,
parallel high-throughput tool for microbial detection, identification and
characterization in natural environments. Genomic method based on microarray
technology may revolutionize the analysis of the structure, function, and kinetics of
microbial community. However, the application of microarray in microbial
community study has just started, and there are many problems preventing microarray
technology to work its full power. The combination of in-situ methods with
microarray may be effective for environmental application, as it is capable of
analyzing the complex community structure of environmental sample (high parallel
capacity of microarray) directly in the field (in-situ study). For Community Genome
Array (CGA), though this novel method is still isolation- dependent for now, there are
ways to make it possible for detecting uncultivated microorganisms in the future, such
as accessing high-molecular weight DNA of microorganisms that are not isolated
through Bacterial Artificial Chromosomes (BACs). So far, CGAs are usually built
with genome DNA from bacteria, but in natural environment, microbial communities
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中南大学本科论文
第 3 章 实验流程
are constituted with bacteria, aechaea and eukaryotes (such as fungi). In the future,
CGA should contain genomic information from bacteria, aechaea and eukaryotes in
order to be more representative, because even in simple systems that contain
relatively fewer kinds of microbial species (such as the AMD system), there has been
report about the existence of aechaea and eukaryotes besides bacteria, let alone
systems in modest natural field. Through CGA fabricated with genomic DNA from
bacteria, aechaea and eukaryotes, we will be able to obtain more comprehensive, and
thus more representative information about the structure of the microbial community
under investigation. In all, various molecular methods have provided useful tool for
research in microbial community because of their sensitivity and accuracy.
Microarray technology has built a novel platform for research in microbial
community because of its obvious advantages in capacity, sensitivity, accuracy and
the potential in giving quantitative information. Although there are still various
problems concerning the experimental system and data analysis, microarray
technology has allowed comprehensive research in function, genetics as well as
regulatory networks and may bring study in microbial community into a new era.
Chapter 3 Materials and experiment
3.1 Materials and instruments used in experiments
Reagents and materials used in experiment are listed in table 3.1
Table 3.1 Reagents and materials used in experiment
Reagents
Manufacturer
(NH4)2SO4
Shantou Xilong Chemical Factory, Guangdong
KCl
Damao Chemical Factory, Tianjin
Chenfu Chemical Factory, Tianjin
MgSO4•7H2O
Shantou Xianhua Chemical Factory, Guangdong
FeSO4•7H2O
K2HPO4
Shiyi Chemical Reagent Co., Ltd. Shanghai
Ca(NO3)2
Chenfu Chemical Factory, Tianjin
Formamide
Damao Chemical Factory, Tianjin
Lysozyme
Amersco Inc. USA
Protainase K
Merck Inc. USA
Rnase
Sigma Inc. USA
Dimethyl Sulphoxide (DMSO)
Shantou Xianhua Chemical Factory, Guangdong
Double distilled water
/
H2SO4
Shantou Xilong Chemical Factory, Guangdong
NaOH
Shiyi Chemical Reagent Co., Ltd. Shanghai
DNA Marker
λDNA/Hind III Lot#F4324 Tiangen Inc. Beijing
DNA Marker
λDNA/Hind III Lot#E3912 Tianweishidai Inc. Beijing
Random Primer
Invitrogen Inc. USA
dNTP
Invitrogen Inc. USA
Klenow
Invitrogen Inc. USA
Cy5dye
Amersham Bioscience Limited UK
Herring Sperm DNA
Invitrogen Inc. USA
5%SDS
Shiyi Chemical Reagent Co., Ltd. Shanghai
Laboratory Film
Pechiney Plastic Racking Company USA
Array Slides
Corming Inc. USA
Conical flask
Tianheng laboratory Instruments Company, Ningbo
Instruments used in the whole experiment process are listed in table 3.2
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中南大学本科论文
第 3 章 实验流程
Table 3.2 Instruments in experiment process
Instruments
Rocking Incubator
Centrifugator
Constant Temperature Water Incubator
Electrophoresis system
Gel Image analysis system
Optical Microscopes
Concentrator
Super Clean Benches
PCR equipment
Scanner
Model
SKY 1102 C
5804 R
SC-15
DYY-2C
T230
CX 31
5301
CBV 1500A
576BR 0958
Genepix Personel
4100A
Manufacturer
Sukun
Eppendorf Inc. Germany
Ningbo City, China
Liuyi Company China
UVitec Inc. USA
Olympus Inc. Japan
Eppendorf Inc. Germany
Regal Inc. Shanghai China
Bio-Rad Inc. USA
Axon instruments Inc. USA
3.2 Bacteria cultivation and the extraction of genomic DNA
In order to fabricate Community Genome Array for the detection and evaluation of
microbial community in bioleaching systems, 27 strains of bacteria Acidiphilum,
Acidithiobacillous Ferrooxidan, Leptospirillum were isolated beforehand by doctor
Gao et al. All these bacteria are sampled in various domestic mining areas. Table 3.1
introduces the locus information about each of these bacteria from where they were
isolated. All the bacteria are isolated from samples obtained in various mine system
except Acidithiobacillous Ferrooxidan 23270, which is bought from USA and is used
as a modeling bacteria. The DNA of other three bacteria S-1, S-2, and J-1 are offered
by Doctor Gao etc. Among these bacteria, 10 strains of Acidithiobacillous
Ferrooxidan are cultivated in 9k medium ( (NH4)2SO4 (3.0g/L), KCl (0.1g/L),
MgSO4•7H2O (0.1g/L), K2HPO4 (0.5g/L), Ca(NO3)2 (0.01g/L) , the pH value was
adjusted to 2) with the addition of FeSO4 (44.6g/L); 10 strains of Leptospirillum are
cultivated in 9k medium with FeSO4•7H2O (40g/L) and the pH value was adjusted to
1.6; 7 strains of Acidiphilum were cultivated in 9k medium added with glucose (10g/L)
and the pH value is adjusted to 3.0. The inoculation of the bulk of the bacteria
mentioned above has succeeded, but we failed to successfully inoculate some of these
bacteria, which we also denoted in Table 3.1. After placed in constant-temperature
incubator and cultivated for about 3~4 days under 30ºC, the bacteria are collected and
washed with H2SO4 and distilled water in order to reduce the attachment of bacteria to
mineral particles formed during cultivation. Then the genomic DNA was extracted
using the EZ-10 Spin Column Genomic DNA Minipreps Kit Protocol-BS423 (Bio
Basic Inc. USA) and preserved in refrigerator at 4ºC. The extraction processes are
listed as follows:
(1). Sample Preparation for cells grown in suspension
Spin appropriate number of cells (max.5 million cells) at 10000rpm for 5min at room
temperature. Remove supernatant completely and discard. Wash the cell pellet twice
with PBS and resuspend in 200ul cold TE buffer, continue the procedure with Step2.
(2). Add 400ul Digestion Solution to 200ul sample from step 1 Mix well. Add 20ul
lysozyme and 40~50ulRNase(10mg/ml), place the system for 5min in room
temperature. Add 20ul proteinase K solution (1ug/ul), and incubate at 55ºC for 10
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中南大学本科论文
第 3 章 实验流程
minutes.
Table 3.3 Locations from where the isolated bacteria are sampled (bacteria are named after
their sample location instead of taxonomic name for the convenience of research)
Genus
Acidithiobacillous
Ferrooxidan
Leptospirillum
Acidiphilium
Bacteria
Isolation
F1
F2
F3
F4
F5
F6
BY
TK
YN
23270
YN4
DY
J5
LY
BY
GXLF
JE
Y17
FTH
ZTS
DBS1
DBS2
DBS3
S-1
J-1
S-2
DBS4
DX1-1
DX5
DX6
Sample Location
Chengmenshan Copper Mine (Jiangxi )
Qixiashan Vanadium Mine (Nanjing)
Dabaoshan Copper Mine (Guangdong)
Yangtaowu Reservoir (Jiangxi)
Liuyang Mine (Hunan)
Dexing Copper Mine (Jiangxi)
Baiyin Copper Mine (Gansu)
Gaofeng Copper Mine (Guangxi)
Yunnan Province
Bought from USA
Yunnan Province
Daye Hubei Province
Dexing Copper Mine (Jiangxi)
Liuyang Hunan Province
Baiyin Yunan Province
Guangxi Province
Dexing Copper Mine (Jiangxi)
Yunnan Province
Dexing Copper Mine (Jiangxi)
Zhongtiao Mountain
Dabaoshan Copper Mine (Guangdong)
Dabaoshan Copper Mine (Guangdong)
Dabaoshan Copper Mine (Guangdong)
DNA offered by others
DNA offered by others
DNA offered by others
Dabaoshan Copper Mine (Guangdong)
Dexing Copper Mine (Jiangxi)
Dexing Copper Mine (Jiangxi)
Dexing Copper Mine (Jiangxi)
Inoculation
Success
Success
Success
Success
Failed
Success
Success
Success
Success
Success
Success
Success
Success
Success
Success
Success
Success
Success
Success
Success
Success
Success
Failed
/
/
/
Success
Failed
Failed
Success
(3). Add 260ul of anhydrous ethanol, and mix well. Apply the mixture to column that
is in a 2.0 ml collection tube. Spin at 10000 rpm for 1 minute.
(4). Discard the flow-through in the collection tube. Add 500ul of Wash Solution, and
spin at 10000 rpm for 1 minute.
(5). Repeat washing step 4.
(6). Discard flow-through. Spin at 12000 rpm for an additional minute to remove
residual amount of Wash Solution.
(7). Place the column into a clean 1.5 ml microfuge tube. Add 30-50ul Elution Buffer
into the center part of membrane in the column. Incubate the tube at 37 or 50.C for 2
minutes. Incubate at 37 or 50ºC could increase recovery yield.
11
中南大学本科论文
第 3 章 实验流程
(8). Spin at 10000 rpm for 1 minute to elute DNA from the column.
(9). Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent of
50ug). Assess genomic DNA quality through 0.7% agarose gel electrophoresis.
Isolated genomic DNA should not contain RNA. This kit is capable of extracting
DNA longer than 50 kb, as it claims in its instruction book, but due to various factors
such as flux in incubation time and temperature in our experiment, the DNA
fragments we obtain are generally about 20kb in length. The length and the
concentration of the DNA obtained were examined through agarose gel
electrophoresis. Results showed that the DNA acquired were about 23kb in length,
which is qualified for the fabrication of a CGA. DNA concentration was adjusted to
about 100ng/µl (±20ng/µl) through condensation and dilution, as the fabrication of
target do not require stringent amount of sample concentration. Besides, reports from
Jizhong Zhou et al. suggest that the combination of DNA to the chip will saturate
when the DNA concentration is more than 200ng/µl [7].
3.3 CGA fabrication
3.3.1 Sample Pretreatment: target DNA preparation
Target DNA refers to the genomic DNA we extracted from various pure culture of
bacteria and will be printed on the surface of the slides and will combine with the
active sites on the surface of the slides. It will then be used to detect probe DNA
through hybridization experiments. After adjusting DNA concentration, the following
manipulations has been done to produce DNA molecule suitable for target printing:
(1). Add 5ul of each DNA sample (24 DNA samples in total) successively into a
384-pore plate
(2). Add 5ul Dimethyl Sulphoxide (DMSO) to each of the DNA sample.
(2). Centrifuge the mixture shortly at 2000 rpm.
(3). Cover the plate tightly with laboratory film; place the plate in an incubator (30ºC
180rpm) for 30min to thoroughly mix the solution.
(4). Centrifuge the mixture shortly at 2000 rpm for another 1 minute.
3.3.2 Microarray fabrication
In total 8 slides, with 1 array that contains 10(2*5) identical 4*7 subarrays on each
one of them, were fabricated in uniform in order to produce statistical data. TE buffer
is used as the negative control. The position information about the samples planted on
the array is listed in table 3.4:
Table 3.4 Community Genome Array: subarray structure (TE buffer has been used as
negative control (blank) in all successive experiments)
Subrow 1
Subrow 2
Subrow 3
Subrow 4
Subc
ol 1
blank
blank
blank
blank
Subcol 2
Subcol 3
Subcol 4
Subcol 5
Subcol 6
Subcol 7
DBS2(A.p)
S-1(A.p)
J-1(A.p)
S-2(A.p)
ZTS(L)
JE(L)
LY(L)
DX6(A.p)
YN(L)
J5(L)
GXLF(L)
DY(L)
TK(At.f)
FTH(L)
BY(L)
Y17(L)
F4(At.f)
F6(At.f)
YN(At.f)
BY (At.f)
23270
F1(At.f)
F2(At.f)
F3(At.f)
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中南大学本科论文
第 3 章 实验流程
The printing work is done in about 1 hour plus 50 minutes through OmniGrid Accent
Microarrayer (GeneMachines Inc. USA) and its corresponding software. After printed
onto the slides, the negative phosphate group on the skeleton of DNA molecule will
combine with the positive (NH3)+ group because of electrostatic interaction. Thus the
DNA molecule will lie on the surface of the slides, while in aldehyde-silane coated
surface the DNA molecule will erect on the surface because of the covalent binding.
This process should be defined as absorption, as the binding of DNA molecule to
(NH3)+ group is nonspecific. The reaction is illustrated in Figure 3.1[10] below:
Figure 3.1 Combination of target DNA to amide group (excerpted from reference 10)
This figure illustrates the combination of target DNA to the active sites
on the surface of the slides. The interaction between the negative
phosphate group of the DNA molecule and the positive NH3+ group
makes them combine and thus the target is anchored on the surface of the
slide.
The slides were placed inside the Microarrayer for about 15 hours to stabilize the
combination. Afterward, all the 8 slides were put into UV Crosslinker Model CL-1000
(UVP Inc. USA) in order to perform crosslinking. This part of manipulation is done
for two purposes: one is to further stabilize the combination of DNA fragment with
the amidogen on the surface of the slides, the other is to block all other blank active
positions on the surface of the slides to prevent of false signal brought by the
hybridization of probe DNA to unblocked active site. During this process, the slides
received irradiation with energy of 600,000µJ/cm2 for about 2 minutes. Then the
slides were taken out and cautiously put inside special plastic box and stored at room
temperature for later on experiments.
3.4 Hybridization of CGA for examining its sensitivity, specificity and
quantitativity potential.
In order to test the overall performance of the CGA fabricated, the sensitivity,
specificity and quantitative potential needs to be carefully examined. Among the three
parameters, the sensitivity refers to the lower threshold of the amount of DNA probe
that can be detected through our microarray; specificity refers to the extent to which
hybridization between probe and target took place, if species with lower than 70%
homologous can hybridize with the target, the microarray is limited in performing
highly specific detection; the quantitative potential of a microarray refers to the
existence of linear relationship between probe concentration and the intensity of
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中南大学本科论文
第 3 章 实验流程
hybridization signal. For examining performance of the CGA, we have to conduct just
one hybridization experiment. But at first, the probe must be labeled beforehand.
3.4.1 Fluorescent labeling of Probe
Genomic DNA from bacteria TK of species Acidithiobacillous Ferrooxidan is
selected to test the capability of the chip we fabricated. The relative quantitation of
sample DNA concentration is done through electrophoresis and the corresponding
image analysis system. DNA marker (longest fragment 23kb, 477 ng/µl) is used as
the control. 4µl of the TK sample is diluted to 1×, 0.1×, 0.01×, and 0.001×
respectively to form the concentration gradient. Then the probes are dyed through
direct labeling with the following procedure:
(1). Prepare Mix 1(35µl for each tube)
Random Primer
20µl
gDNA (0.5-2.0µg)
5µl
ddH2O
10µl
(2). Add 35µl of Mix 1 to each tube.
(3). Incubate the system at 98°C for 5 minutes.
(4). Quickly cool down on ice and centrifuge for 10 seconds.
(5). Add dNTP (5mM dA/G/CTP, 2.5mM dTTP) 1.0µl, Klenow 2.0µl, Cy5dye 0.5µl
and ddH2O 11.5µl, mix up and incubate the system at 37°C for 3 hours, quickly cool
down on ice.
(6). Add 1N NaOH 2.5µl and incubate at 37°C for 10 minutes.
(7). Add 1N HCl 2.5µl to neutralize the above solution.
(8). Add Tris-HCl (pH 7.0-7.5) 5.0µl.
(9). Transfer the solution into an absorption column, add 600µl Binding Buffer
(10). Centrifuge at 12,000rpm for 30 seconds.
(11). Add 500µl of Washing Buffer, centrifuge at 12,000rpm for 30 seconds, and then
wash again.
(12). Add 50µl of Buffer EB, transfer the column into a new centrifuge pipe (1.5ml),
incubate at room temperature for 2 minutes, centrifuge at 12,000rpm for 30 seconds.
(13). Condense the solution in vacuum concentrator for about 20 minutes.
3.4.2 Hybridization of labeled probe to the microarray
We have fabricated 8 pieces of CGA in total, among them 4 were tested here to
produce statistical data, that is, the data produced by 40 dots of the identical strain (in
total 24*40=960) were collected and analyzed. Before hybridization, the slides to be
tested are taken out and boiled in distilled water for 2 minutes and washed in 95%
ethanol. The hybridization procedure is as follows:
(1). Add formamide 15µl and ddH2O 5.6µl into a PCR tube; the total volume of the
system is 20.6µl.
(2). Solute the fluorescent labeled DNA in the solution above.
(3). Add the following reagents into the PCR pipe:
20×SSC
5.04µl
Herring Sperm DNA (10µg/µl)
2.4µl
5% SDS
2µl
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中南大学本科论文
第 2 章 综述
Thus, the total volume of the system is 30µl.
(4). Place the above mixture inside the Bio-Rad PCR equipment and incubate at 95°C
for 5 minutes. Cool down the system to 60°C.
(5). Place 4 pieces of the CGA we fabricated inside the UV Crosslinker under
600,000µJ/cm2 for 2 minutes, then place the chips in the Bio-Rad PCR equipment,
incubate at 60°C for 5 minutes.
(6). Transfer the above solution on the surface of the slides; cover it with cover glass,
then add one drop of sterile water to two sides of the hybridization box. Close the
hybridization box tight and place it in the water incubator, hybridize at 55°C
overnight.
(7). Washing after hybridization. (Note that the water used for washing should be
about 50°C).
Washing step 1:
Put the chips inside a clean beaker, add 93ml distilled water, 5ml 20×SSC and 2ml of
10×SDS, shake the beaker slightly to move off the cover glass, wash for 5 minutes,
discard the fluid.
Washing step 2:
Add 97.5ml distilled water, 0.5ml 20×SSC and 2ml of 10×SDS; wash for 10 minutes,
discard the fluid.
Washing step 3:
Add 99.5ml distilled water and 0.5ml 20×SSC, and wash for 30 seconds.
(8) Take out the chips carefully; dry the chips using a blower, then the chips can be
scanned for result check.
3.3.3 Scanning of hybridization result
The hybridization result is scanned using scanner Genepix Personel 4100A (Axon
instruments Inc. USA) and its corresponding software: Genepix Pro 6.0. Note that the
positive surface (surface planted target) of the chips should be adown.
Chapter 4 Results and discussion
The experiments above have produced a series of data. In this part of the assay, the
analysis work is depicted, the results are shown and discussion is made.
4.1 The quantitation of DNA probe concentration.
The quantitation of DNA probe concentration proved to be intractable. Although UV
Spectrophotometer has provided strategy for detecting the concentration of DNA
sample, its capacity in detecting trace amount of DNA is limited. Because the total
volume of DNA we extracted is often less than 100µl, there isn’t enough DNA
sample for us to conduct UV absorption experiment. Instead, we find a way to
relatively quantify sample DNA concentration through agarose gel electrophoresis
and the corresponding analyzing software. Figure 4.1 is the picture of electrophoresis
result of bacteria TK, the parameters are used directly as in the software and
explanations are given according to the instruction book of the software, here we
select parameter ‘volume’ for our work to relatively quantify the concentration of
DNA probe.
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中南大学本科论文
第 2 章 综述
Figure 4.1 Electrophoresis result of TK and Marker
The gel image analysis system software is able to detect the total signal
intensity-which is defined as ‘volume’. According to the detection result, the volume
of the first band of Marker is 99049, and the volume of our sample TK is 119927.
Both Marker and TK are sampled 4µl to run electrophoresis. The signal intensity is
proportional to sample DNA concentration, the Marker we use here has labeled its
concentration of each band (477ng/5µl for the first 23120bp band). Thus, the
DNA concentration of our sample TK can be estimated through the following
equation (X represents the DNA concentration of sample TK):
4  477 / 5 99049

4 
119927
X=115.5ng/µl
Thus, the concentration of bacteria TK is about 115.5ng/µl.
4.2 Surface scanning of CGA after hybridization
After hybridization, the results are checked through scanner Genepix Personel 4100A,
the image obtained are shown in Figure 4.1:
A (1×)
B(0.1×)
C(0.01×)
D(0.001×)
Sample DNA concentration reduce following the direction of the arrow
Figure 4.2 Hybridization result through scanning
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中南大学本科论文
第 4 章 数据处理与结果讨论
The four pictures presented in the last page shows the fluorescent signal
after hybridization the CGA with DNA of bacteria TK, which are in
gradient1×, 0.1×, 0.01×, and 0.001×. Chip A is hybridized with 1×
TK DNA; chip B is hybridized with 0.1×TK DNA; chip C is
hybridized with 0.01×TK DNA, and chip D is hybridized with
0.001×TK DNA. The signal intensity can be easily
differentiated among each other, and chip D, which is
hybridized to 0.001×TK DNA (about 0.11ng of DNA), still
shows clear signal and hybridization specificity.
For successive test of the specificity and quantitative potential, the images shown in
Figure 4.2 were modified through the software Genepix Pro 6.0 and are shown below
in Figure 4.3:
One subarray
A (1×)
B(0.1×)
C(0.01×)
D(0.001×)
Figure 4.3 Modified images of picture A, B, C and D
Modified images of scanning result. The background (color blue) and
interference signals are automatically subtracted through the software
Genepix Pro 6.0. Thus, the contrast of spots that have hybridization signal to
spots that no hybridization has taken place is largely enhanced. For the
quantitation of hybridization signal, image modify is also necessary as the
original images prove to be too coarse to provide information accurate
enough. The images above also present clear trend of change in hybridization
signal intensity from left to right.
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中南大学本科论文
第 4 章 数据处理与结果讨论
4.3 CGA capability evaluation
4.3.1 Evaluation of CGA sensitivity
The sensitivity of the CGA is depicted in Table 4.1
Table 4.1 Result of hybridization signal (positive or negative) under different concentration
of probe DNA
Probe
Concentration
Hybridization Signal
(115ng)
Positive
Negative


(11.5ng)
(1.15ng)
(0.011ng)






The result shows that the CGA we fabricated is capable of detecting extremely small
amount of DNA. In Figure 4.1, from left to right the pictures present the hybridization
results of CGA to sample DNA in concentration gradient 115ng, 11.5ng, 1.15ng and
0.115ng. According to Figure 4.1 picture D, 0.115ng of sample DNA gives clear
fluorescent signal, which means the CGA fabricated is capable of detecting 0.115ng
DNA sample. Note that the surface where the hybridization box placed should be
horizontal, thus the thickness of the fluid between the chip and the cover glass will be
uniform, which guarantees the uniformity of probe DNA distribution. The capability
of the lower limitation of detection capability may still been underestimated because
of the lack of more diluted gradient. But considering environmental application, the
sensitivity of the CGA may not be as high as the experiments prove here, as in the
experiment we use the DNA from a single pure culture. When utilizing this CGA to
detect DNA in mixed natural sample, the sensitivity of the CGA should be lower
because of the interference of various kinds of DNA molecule and chemical
compounds existing in the solution. After all, in terms of testing the sensitivity of the
CGA, we reach the conclusion that the CGA we fabricated is capable of detecting
DNA sample as little as 0.11ng.
4.3.2 Evaluation of CGA specificity
As reported, the specificity of CGA is to differentiate species, and under stringent
conditions it may be possible for the CGA to distinguish tell different strains within a
species. The hybridization condition is 50% formamide at temperature 55°C, the
whole hybridization process lasted for about 13 hours. After hybridization, the chip
are taken our from the hybridization box and carefully washed as described in chapter
3. In order to examine the hybridization result more clearly. We need to refer to Figure
4.2 for more contrasted information. Cross hybridization were observed in strains
DBS2, S-1 and J-1. The reason for this phenomenon is discussed later. Despite the
cross hybridization, hybridization signals are obtained in target 23270, F1, F2, F3, F4,
F6, YN, and BY (At.f). All of the targets listed above are Acidithiobacillous
Ferroxidans. Ten strains of bacteria Leptospirillum showed no hybridization signal.
Thus, the resolution of hybridization of the CGA we fabricated is species, as the
hybridization result shows that the CGA cannot successfully tell different strains of
the same species Acidithiobacillous Ferroxidan. Table 4.2 shows the result of
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中南大学本科论文
第 2 章 综述
hybridization specificity.
Table 4.2 Hybridization result of each of the target in a subarray
Subrow
1
Subrow
2
Subcol 1
Blank

Blank

Subcol 2
DBS2(A.p)

S-1 (A.p)

Subcol 3
ZTS(L)

JE (L)

Subrow
3
Blank

J-1 (A.p)

LY (L)

Subrow
4
Blank

S-2 (A.p)

DX6(A.p)

Subcol 4
YN (L)

J5 (L)

GXLF
(L)

DY (L)

Subcol 5
TK (At.f)

FTH(L)

Subcol 6
F4 (At.f)

F6 (At.f)

Subcol 7
23270(At.f)

F1 (At.f)

BY(L)

YN (At.f)

F2 (At.f)

Y17(L)

BY (At.f)

F3 (At.f)

Those labeled ‘’ own detectable hybridization signal, while those labeled ‘’ have no
hybridization signal.
Explanation of the hybridization signal from target DBS2, S-1, and J-1 should be
made, as they are claimed to be Acidiphilium. In theory, bacteria strains of
Acidiphilium should not be hybridized with DNA from bacteria of Acidithiobacillous
Ferroxidan, as the target DNA are the genomic DNA that are longer than 20kb (about
23 kb), DNA molecule as long as that should be well enough to ensure the
hybridization specificity. One possible reason for the signal here is that the
Acidiphilium S-1, J-1 and DBS2 are not pure cultures, that is, the target prepared
using DNA of the three bacteria above contains DNA molecule of Acidithiobacillous
Ferroxidans. Maybe the isolation of these bacteria failed to obtain a pure culture.
Evidence for the hypothesis here is that there has been report suggesting that bacteria
Acidiphilium and Acidithiobacillous Ferroxidans are closely related in environment,
their metabolic functions promotes each other, thus it is unlikely that the pure
isolation of Acidiphilium could be obtained through conventional isolation strategy.
So the signal detected from bacteria S-1, J-1 and DBS2 may still be the result of
hybridization of our sample probe with Acidithiobacillous Ferroxidans DNA that are
extracted simultaneously with the Acidiphilium in the same culture and planted onto
the surface of the chip.
4.3.3 Evaluation of quantitative potential
In order to evaluate the quantitative potential of the CGA fabricated, the signal
intensity of hybridization result image is obtained through software Genepix Pro 6.0.
The microarray A, B, C and D were hybridized with probe concentration 115ng/µl,
11.5ng/µl, 1.15ng/µl and 0.115ng/µl, respectively. The data of four chips are collected
and sorted out respectively. The comparison of data is made between the same
bacteria species hybridized at different probe concentration. That is, we collected the
data lengthways and the data is compared transverse to produce reliable and
intuitionistic result.
There are 10 identical subarrays on each slide, that is, each of the target has ten
replicates in order to produce statistical data (though the data that departed too much
from the average value are neglected). We calculated the average signal intensity of
each bacteria strain and the result is listed in Table 4.3: Besides, standard deviance
and the log value of each of the signal intensity are also calculated; the log value of
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中南大学本科论文
第 4 章 数据处理与结果讨论
the average signal intensity is obtained for the purpose of successive construction
work.
Table 4.3 Signal intensity of each bacteria strain
Column Row
Name
Average
Average Intensity Average Intensity Average Intensity
Intensity for
for chip A
for chip B
for chip C
chip D
1
1
23270
9930.6
7466.875
1940.5
182.75
2
1
F1(At.f)
13157.3
10373.625
3736.11
274.625
3
1
F2(At.f)
11134.78
8081.875
2422.125
242.625
4
1
F3(At.f)
17573.56
15846.125
5219.375
548.5
1
2
F4(At.f)
9014
6961.75
2063.9
176
2
2
F6(At.f)
9698.44
7748.125
2264.4
185
3
2
YN(At.f)
11809.33
10460.75
4402.89
544.25
4
2
BY (At.f)
14193.78
12027.75
4014.4
345.625
1
3
TK(At.f)
6881.11
4939.875
1404
117.125
2
3
FTH(L)
86.33
41
15.8
6.875
3
3
BY(L)
198.5
122.86
37.4
10.375
4
3
Y17(L)
44.6
35
12.5
8.375
1
4
YN(L)
47.4
28.7
12.8
5.5
2
4
J5(L)
24.2
20.3
8.2
9
3
4
GXLF(L)
19.8
18
7.6
5.9
4
4
DY(L)
31.4
26.7
12.3
8
1
5
ZTS(L)
10.6
12.8
9.9
4.4
2
5
JE(L)
22.5
19
5.2
6.9
3
5
LY(L)
15.6
15.4
5.3
5.9
4
5
DX6(A.p)
822.4
690.9
225.5
16.25
1
6
DBS2(A.p)
10278.22
8278.143
2274.7
158.375
2
6
S-1(A.p)
12041.67
7796.9
2125.7
2068.78
3
6
J-1(A.p)
4205.78
3174.8
849.5
1070.75
4
6
S-2(A.p)
89.3
20.3
10.8
7.375
1
7
blank
11.3
9.2
6.2
6.2
2
7
blank
5.3
5.9
5.4
4.9
3
7
blank
6.7
6.2
5
3.1
4
7
blank
4.3
3.7
3.8
3.8
Through the data listed above, we can easily discover that the signal intensity reduce
as the concentration of probe DNA reduce. This table also can be used to demonstrate
the specificity of CGA, as the signal intensity for Leptospirillum is magnificently
fewer than that of Acidithiobacillous Ferroxidans (the signal for Acidiphilium is
explained in 4.3.2). After obtaining these data, we use the log value of hybridization
signal intensity and the log value of probe DNA amount to from data pairs (X, Y).
These spots are then placed in coordinates for the purpose of checking CGA
quantitative potential. Because there are 9 Acidithiobacillous Ferroxidans on the
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中南大学本科论文
第 4 章 数据处理与结果讨论
4.2
4.4
4.0
4.2
3.8
4.0
3.6
3.8
Log Signal Intensity
Log Siganl Intensity
chip, and the probe DNA is from strain TK, which is also within this species, all 9
series of data pairs are sorted and portrayed from Figure 4.4 to Figure 4-12:
3.4
3.2
3.0
2.8
3.6
3.4
3.2
3.0
2.6
2.8
2.4
2.6
2.2
2.4
2.0
2.2
-1.0
-0.5
0.0
0.5
1.0
Log DNA amount/ng
(1) 23270
2
r =0.91 p<0.05
1.5
2.0
-1.0
2.5
-0.5
0.0
0.5
1.0
Log DNA amount/ng
(2) F1
1.5
2.0
2.5
1.5
2.0
2.5
2
r =0.87 p<0.07
Figure 4.4
Figure 4.5
4.8
4.4
4.6
4.2
4.4
4.2
3.8
Log Signal Intensity
Log Signal Intensity
4.0
3.6
3.4
3.2
3.0
4.0
3.8
3.6
3.4
3.2
2.8
3.0
2.6
2.8
2.4
2.6
2.2
-1.0
-0.5
0.0
0.5
1.0
Log DNA amount/ng
(3) F2
1.5
2.0
2.5
2
2.4
-1.0
-0.5
0.0
0.5
1.0
Log DNA amount/ng
(4) F3
2
r =0.91 p<0.05
r =0.89 p<0.06
Figure 4.6
Figure 4.7
21
第 4 章 数据处理与结果讨论
4.2
4.2
4.0
4.0
3.8
3.8
3.6
Log Signal Intensity
Log Signal Intensity
中南大学本科论文
3.4
3.2
3.0
3.6
3.4
3.2
3.0
2.8
2.8
2.6
2.6
2.4
2.4
2.2
2.2
2.0
2.0
-1.0
-0.5
0.0
0.5
1.0
Log DNA amount/ng
(5) F4
1.5
2.0
-1.0
2.5
-0.5
2
r =0.89 p<0.06
Figure 4.8
0.0
0.5
1.0
Log DNA amount/ng
(6) F6
2
r =0.89 p<0.06
1.5
2.0
2.5
2.0
2.5
Figure 4.9
4.6
4.4
4.4
4.2
4.2
4.0
3.8
Log Signal Intensity
Log Signal Intensity
4.0
3.6
3.4
3.2
3.8
3.6
3.4
3.2
3.0
3.0
2.8
2.6
2.8
2.4
2.6
2.2
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
-1.0
2.5
Log DNA amount/ng
(7) YN
2
r =0.86 p<0.08
-0.5
0.0
0.5
1.0
Log DNA amount/ng
(8) BY
2
r =0.87 p<0.07
Figure 4.10
Figure 4.11
22
1.5
中南大学本科论文
第 5 章 结论
4.0
3.8
Log Signal Intensity
3.6
3.4
3.2
3.0
2.8
2.6
2.4
2.2
2.0
1.8
-1.0
-0.5
1.0
0.5
0.0
Log DNA amount/ng
(9) TK
1.5
2.0
2.5
2
r =0.91 p<0.05
Figure 4.12
Figures above shows the relationship of the intensity of hybridization signal and the
corresponding probe DNA concentration. The log value of signal intensity data is
calculated and the standard deviances are shown in the figures above. Although some
of the point showed remarkable deviance, the trends of increase in signal intensity
with the increase of probe concentration of the 9 strains of bacteria are nearly
identical. Linear regression results suggest that the CGA we fabricated is potentially
quantitative. Figure 4.11 shows the linear regression result of bacteria TK hybridized
with DNA of itself (r2=0.91 p<0.05) and thus is the most representative one in
demonstrating the quantitative potential of the CGA to detect pure DNA sample.
Chapter 5 Conclusion
The capability of the CGA we fabricated is examined in terms of sensitivity,
specificity and quantitative potential. Analysis of the results suggests that the CGA
fabricated is capable of detecting sample DNA as little as 0.11ng while still
possessing good specificity. All of the hybridization of target DNA with DNA from
bacteria TK are in the species Acidithiobacillous Ferrooxidan except for J-1, S-1, and
DBS-2, suggesting that the CGA fabricated is capable of differentiating bacteria in the
species level. The hybridization signal observed in target J-1, S-1, and DBS-2 may
because of the culture from where the target DNA were extracted is not pure itself, as
there have been report claiming that bacteria Acidiphilium and Acidithiobacillous
Ferrooxidan are closely related in natural environment and it is extremely difficult to
isolate the pure culture of Acidiphilium. Thus, the hybridization signal observed
between probe DNA and J-1, S-1, and DBS-2 should still be brought by hybridization
23
中南大学本科论文
第 5 章 结论
occurred between the probe DNA and the DNA of Acidithiobacillous Ferrooxidan
mixed in the above Acidiphilium bacteria. More stringent hybridization condition
(such as higher hybridization temperature and longer washing time) may further
enhance the resolution of the CGA and makes it possible for the CGA to detect
different strains of bacteria. The fact that the target DNA of bacteria TK did not prove
to be show the strongest fluorescent signal as we expected may because of the lower
DNA concentration of the TK target printed on the chip. Dying result using Sybr
Green (Figure 5.1) proves that the target of TK possess fewer amount of DNA than
other bacteria target, the picture also explains the strong signal intensity of target YN,
because the target itself contains remarkably more amount of DNA.
Target TK
Target YN
Figure 5.1 Target DNA concentration check
Target DNA concentration is checked through dying using Sybr Green
(trade name), spots with light yellow color indicate fewer DNA and
spots in brown or red indicate more amount of DNA. The target DNA
of TK is lower than the other bacteria of Acidithiobacillous
Ferrooxidan in concentration. Bacteria target YN possesses the highest
DNA concentration. This explains why YN is always the one that
shows strongest hybridization signal.
The above result suggests that the quantitation of DNA concentration for the
preparation of target is of magnificent importance. Hybridization result will be more
representative and reliable when the target DNA concentrations are uniform.
Considering quantitative potential, the analysis of hybridization signal intensity data
produced by different probe DNA concentration has provided evidence for the
quantitative potential of the CGA. In total, the construction of CGA is successful:
high sensitivity has enhanced its capability to detect minor species in environmental
sample. Species level resolution will remarkably reduce cross-hybridization, and the
resolution still have the potential to be improved to strain level by changing
hybridization conditions, more stringent hybridization will wash off the probe
combined to the less homologous target while correct hybridization will stay because
the combination is more stable. The quantitativity of the CGA can provide useful in
determining the relative abundance of detectable species in environmental sample.
The quantitation potential of CGA may be further determined through analysis of
more parallel data, that is, we can obtain probe DNA concentration of 300ng/µl,
250ng/µl, 200ng/µl, 150ng/µl, 100ng/µl, 50ng/µl, 20ng/µl, and 10ng/µl through
condensation and dilution to form a more complex concentration gradient. Thus, we
will be able to obtain a lot more detailed data about the influence of probe DNA
24
中南大学本科论文
致谢及参考文献
concentration on hybridization signal intensity. One weakness of the CGA fabricated
is the lack of richness of species diversity, but this could be compensated by later on
isolation and cultivation of various bacteria. The bacteria DNA we extracted are
stored at –20ºC, thus these sample can still be used to fabricate new chips, once new
isolation or cultivation is obtained, its genomic DNA can instantly be applied in CGA
fabricating, the richness of target genomic DNA will be expanded continually. Besides,
methods such as utilizing BACs to obtain genomic DNA of uncultivated species have
offered new strategy in CGA fabrication. The limitations of CGA application will be
impaired and CGA will work its full power in research toward various environmental
applications.
Acknowledgement
The experimenting process, including the inoculation and cultivation of bacteria,
DNA extraction and concentration adjusting, microarray fabrication and successive
testing and examining, lasted for more than four months. The authur wish to thank
professor Liu xueduan for his continous guiding throughout the whole period and
docter Gao Jian, master Zhang Yanfei for offering the bacteria isolations. I sincerely
thank graduate student Chen Qijiong and other graduate students for the cooperation
in the whole experimenting process. Besides, teachers in our laboratory have also
helped me a lot in inquiring and instructing experimental instruments manipulation. In
the end, I also thank my classmates for sharing a happy and meaningful time in the
last semester of undergraduate year in Central South University.
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