U.S. patent application number 14/345671 was filed with the patent office on 2014-08-14 for method and kit for constructing plasma dna sequencing library.
This patent application is currently assigned to BERRY GENOMICS CO., LTD.. The applicant listed for this patent is Di Chen, Yang Gao, Yanbin Shi, Feng Tian, Jianguang Zhang. Invention is credited to Di Chen, Yang Gao, Yanbin Shi, Feng Tian, Jianguang Zhang.
Application Number | 20140228256 14/345671 |
Document ID | / |
Family ID | 48781020 |
Filed Date | 2014-08-14 |
United States Patent
Application |
20140228256 |
Kind Code |
A1 |
Zhang; Jianguang ; et
al. |
August 14, 2014 |
METHOD AND KIT FOR CONSTRUCTING PLASMA DNA SEQUENCING LIBRARY
Abstract
The disclosure relates a method and a kit for constructing a
plasma Deoxyribonucleic acid (DNA) sequencing library. The method
provided by the disclosure includes: extracting a plasma DNA;
making the plasma DNA ligate to a sequencing linker, and purifying
a ligation product; performing Polymerase Chain Reaction (PCR)
amplification for the purified ligation product, purifying the PCR
amplification product, and obtaining the plasma DNA sequencing
library, wherein, the method does not include the step of
performing 5'-terminus phosphorylation for the plasma DNA. The kit
provided by the disclosure includes: a reagent which ligates a
plasma DNA to a sequencing linker, including the sequencing linker,
a ligase and a ligation buffer; and reagents and instruments for
purifying the ligation product; a reagent which performs PCR
amplification for a purified ligation product, and reagents and
instruments for purifying the PCR amplification product; wherein,
the kit does not include the reagent which performs 5'-terminus
phosphorylation for the plasma DNA. The disclosure simplifies the
construction flow of a plasma DNA sequencing library, simplifies
the experimental procedures, and makes the construction of the
plasma sample library have lower cost, higher efficiency and faster
speed, and is convenient for large-scale application.
Inventors: |
Zhang; Jianguang; (Beijing,
CN) ; Gao; Yang; (Beijing, CN) ; Shi;
Yanbin; (Beijing, CN) ; Chen; Di; (Beijing,
CN) ; Tian; Feng; (Beijing, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Zhang; Jianguang
Gao; Yang
Shi; Yanbin
Chen; Di
Tian; Feng |
Beijing
Beijing
Beijing
Beijing
Beijing |
|
CN
CN
CN
CN
CN |
|
|
Assignee: |
BERRY GENOMICS CO., LTD.
Beijing
CN
|
Family ID: |
48781020 |
Appl. No.: |
14/345671 |
Filed: |
January 10, 2012 |
PCT Filed: |
January 10, 2012 |
PCT NO: |
PCT/CN2012/070162 |
371 Date: |
March 19, 2014 |
Current U.S.
Class: |
506/26 ; 435/194;
435/6.12 |
Current CPC
Class: |
C12Q 1/6806 20130101;
C12Q 1/6806 20130101; C12N 15/1093 20130101; C12Q 2535/122
20130101; C12Q 2525/191 20130101; C12Q 2521/525 20130101 |
Class at
Publication: |
506/26 ; 435/194;
435/6.12 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method for constructing a plasma Deoxyribonucleic acid (DNA)
sequencing library, comprising: extracting a plasma DNA; ligating
the plasma DNA with a sequencing linker, and purifying a ligation
product; performing Polymerase Chain Reaction (PCR) amplification
for a purified ligation product, purifying a PCR amplification
product, and obtaining the plasma DNA sequencing library; wherein,
the method does not comprise the step of performing 5'-terminus
phosphorylation for the plasma DNA.
2. The method according to claim 1, wherein, ligating the plasma
DNA with the sequencing linker is performed by adopting one or more
ligases selected from the group consisting of: a T4 DNA ligase, a
T4 Ribonucleic Acid (RNA) ligase and a T7 DNA ligase.
3. The method according to claim 1, wherein the sequencing linker
is a single-strand sequencing linker.
4. The method according to claim 1, wherein the sequencing linker
is a double-strand sequencing linker.
5. The method according to claim 4, wherein, after extracting the
plasma DNA, and before ligating the plasma DNA with the sequencing
linker, performing dA-overhang for the extracted plasma DNA, and
purifying a plasma DNA with dA-overhang.
6. The method according to claim 5, whereina the dA-overhang adopts
klenow ex-enzyme, or Taq enzyme, or a combination of klenow
ex-enzyme and Taq enzyme.
7. The method according to claim 4, wherein, after extracting the
plasma DNA, and before ligating the plasma DNA with the sequencing
linker, the method further comprises performing end-filling and
dA-overhang for the extracted plasma DNA.
8. The method according to claim 7, wherein the method comprises
performing the end-filling and dA-overhang in one reaction system,
and then purifying an end-filled and dA-overhanged plasma DNA.
9. The method according to claim 8, wherein T4 DNA polymerase is
adopted for the end-filling, and Taq enzyme is adopted for the
dA-overhang.
10. The method according to claim 7, wherein the method comprises
performing the end-filling, the dA-overhang, and the ligation of
the plasma DNA with the sequencing linker in one reaction
system.
11. The method according to claim 10, wherein T4 DNA polymerase is
adopted for the end-filling, and Taq enzyme is adopted for the
dA-overhang.
12. A kit for constructing a plasma DNA sequencing library,
comprising: a reagent which ligates a plasma DNA to a sequencing
linker, the reagent comprising the sequencing linker, a ligase and
a ligation buffer; reagents and instruments for purifying a
ligation product; a reagent which performs PCR amplification for a
purified ligation product and reagents and instruments for
purifying a PCR amplification product; wherein, the kit does not
comprise a reagent which performs 5'-terminus phosphorylation for
the plasma DNA.
13. The kit according to claim 12, wherein, the ligase is selected
from the group consisting of: T4 DNA ligase, T4 RNA ligase and T7
DNA ligase.
14. The kit according to claim 12, wherein the sequencing linker is
a single-strand sequencing linker.
15. The kit according to claim 12, wherein the sequencing linker is
a double-strand sequencing linker.
16. The kit according to claim 15, wherein the kit further
comprises: a reagent which performs dA-overhang for a extracted
plasma DNA, the reagent comprising Deoxyadenosine Triphosphate
(dATP), an enzyme for dA-overhang, a buffer for dA-overhang; and
reagents and instruments for purifying the plasma DNA with
dA-overhang.
17. The kit according to claim 16, wherein the enzyme for
dA-overhang is klenow ex-enzyme, or Taq enzyme, or a combination of
klenow ex-enzyme and Taq enzyme.
18. The kit according to claim 15, further comprising: a reagent
which performs end-filling and dA-overhang for the extracted plasma
DNA, the reagent comprising an enzyme for end-filling,
Diethyl-Nitrophenyl Thiophosphate (dNTP), a buffer for end-filling,
Deoxyadenosine Triphosphate (dATP), an enzyme for dA-overhang, and
a buffer for dA-overhang; and reagents and instruments for
purifying an end-filled and dA-overhanged plasma DNA.
19. The kit according to claim 15, wherein, the kit further
comprising: a reagent which performs end-filling and dA-overhang
for the extracted plasma DNA in one reaction system, the reagent
comprising an enzyme for end-filling, dNTP, a buffer for
end-filling, dATP, an enzyme for dA-overhang, and a buffer for
dA-overhang; and the reagents and instruments respectively for
purifying an end-filled and dA-overhanged plasma DNA.
20. The kit according to claim 19, wherein, the enzyme for
end-filling is T4 DNA polymerase; and the enzyme for dA-overhang is
Taq enzyme.
21. The kit according to claim 15, the kit further comprising: a
reagent which performs end-filling and dA-overhang for the
extracted plasma DNA, the reagent comprising an enzyme for
end-filling, dNTP, a buffer for end-filling, dATP, an enzyme for
dA-overhang, and a buffer for dA-overhang; wherein the end-filling
and dA-overhang, and the ligation between the plasma DNA and the
sequencing linker, are performed in one reaction system.
22. The kit according to claim 21, wherein, the enzyme for
end-filling is T4 DNA polymerase; and the enzyme for dA-overhang is
Taq enzyme.
Description
TECHNICAL FIELD OF THE APPLICATION
[0001] The disclosure relates to a method and a kit for
constructing a plasma Deoxyribonucleic acid (DNA) sequencing
library, and in particular to a method and a kit for constructing a
plasma DNA sequencing library for the second-generation
high-throughput sequencing.
[0002] Background of the application
[0003] With the progress of science, the traditional Sanger
sequencing cannot fully satisfy the needs of the research; the
genome sequencing needs a sequencing technology which has lower
cost, higher throughput and faster speed, so the second-generation
sequencing technology is emerged at the right moment. The core idea
of the second-generation sequencing technology is sequencing by
synthesis, namely, to determine the DNA sequence by catching the
mark of the newly-synthesized end; the existing technical platform
mainly includes Roche/454 FLX, Illumina/Solexa Genome Analyzer and
Applied Biosystems SOLID system and the like. Taking Illumina
product as an example, the GAII reading length has been developed
to be 100 bases at present from 36 bases in 2008, and the
throughput has been developed to be 240M reads/run at present from
48M reads/run in 2008, the sequencing capacity has been improved by
14 times. By now, each run of HiSeq 2000 can achieve 3 human
genomes and 30.times. coverage sequencing throughput, approximately
300 G/run data, and the processing time on an equipment has been
reduced to 30 min. As the second-generation sequencing technology
becomes more mature, it is rapidly developed to be applied in
clinical research. Studies show that genetic health condition of a
fetus can be judged by sequencing the maternal plasma DNA; and the
early cancer screening can be performed by sequencing the plasma
DNA of test people, thus the second-generation sequencing
technology has a strong prospect for application.
[0004] The plasma DNA is also called circulating DNA, which is the
extracellular DNA in the blood, and of which the length is
approximately tens to hundreds of nucleotides; the plasma DNA can
exist in the form of DNA-protein complex, and also can be free DNA
fragments. Under normal circumstances, the plasma DNA derives from
the DNA which is released by a small amount of aging dead cells. In
a healthy state, the generation and removal of the circulating DNA
are in a dynamic balancing state, and are maintained at a
relatively constant low level. The circulating DNA can reflect the
status of the cell metabolism of the human body, which is an
important indicator of health evaluation. The change of quantity
and quality of the peripheral blood circulating DNA have a close
relationship with various diseases (including tumour, severe
composite trauma, organ transplantation, pregnancy-related
diseases, infectious diseases, organ failure and the like); as a
non-invasive detection indicator, the circulating DNA can possibly
become an important molecular marker for performing early
diagnosis, illness monitoring, therapeutic effect evaluation and
prognosis evaluation of some diseases.
[0005] Since the presence of fetal DNA in maternal plasma was
validated.sup.(3), non-invasive prenatal diagnosis and detection of
fetal chromosomal abnormalities has become a major research
subject. In 2007, Professor Lu Yuming and his colleagues proved
that the ratio of mutation site of the placental specific gene 4 in
maternal plasma Messenger Ribonucleic Acid (RNA) (mRNA) could be
used to judge whether the fetus has chromosome 21# which was a
triploid.sup.(4). The ratio of mutation site is also used for
judging whether the chromosome 18# is triploid.sup.(5). The
limitation of these methods above is that the mutation site is not
common in the crowd, therefore, these methods are only suitable for
a part of crowd. During the same period, digital Polymerase Chain
Reaction (PCR) (dPCR) is used for detecting the fetus with
chromosomal triploidy.sup.(6), (7). The advantage of the dPCR is
that it does not depend on any mutation site; however, the accuracy
of dPCR is insufficient , and also requires a large amount of blood
samples, which increases the difficulty in sampling.
[0006] In recent years, the high-throughput sequencing technologies
which are rapidly developed have solved the problems above. These
technologies include Genome Analyzer.sup.(8) of Illumina Company,
SOLiD.sup.(9) of Life Technologies Company, and Heliscope.sup.(10)
of Helicos Company, by which hundreds of millions or even billions
of sequences can be detected for once. When detecting the DNA in
maternal plasma by these technologies, the change of the chromosome
number of a trace amount of fetal DNA in the plasma can be
detected.sup.(11), (12), (13). But because of high sequencing cost,
the technologies have not yet been widespread used at present.
Meanwhile, the way of detecting the change of local copy number of
the embryonic chromosomes from the maternal blood is an unsolved
problem. There are some advantages to detect the change of copy
number of the fetal chromosomes from maternal plasma by the
high-throughput sequencing; however, the high-throughput sequencing
is expensive and cannot be popularized. Moreover, the Coefficient
of Variation (CV) of sequencing is high, and the accuracy and
stability of detection also need to be improved. And the CV of
sequencing also determines that the method is only suitable for a
few chromosomes, such as chromosome 21# and chromosome 18#, but
unsuitable for detecting the change of partial copy number of the
chromosomes at present.
[0007] The improvement of the sequencing efficiency and the
popularization of multi-sample mixed sequencing demand higher
efficiency in preparing samples, particularly in preparing large
number of clinical samples; however, the present method of
preparing clinical plasma sample pull up to the improvement of the
sequencing capacity. Therefore, the efficiency and cost in
preparing clinical plasma DNA samples for the second-generation
high-throughput sequencing have become the key for high-throughput
sequencing to be popular.
[0008] Essentially, the process of preparing plasma DNA samples for
the second -generation high-throughput sequencing is inserting the
DNA which satisfies the sequencing length into the existing
sequencing vector, namely, ligating the known sequencing linker on
two ends of the DNA to be sequenced. At present, the construction
of the plasma DNA library mainly includes: firstly performing
end--repairing and 5'-terminus phosphorylation for the extracted
plasma DNA, and then performing the main steps, such as
dA-overhang, linker ligation and PCR and the like (FIG. 1),
wherein, the purification step needs to be implemented in each of
the steps above. Such construction method of the plasma DNA
sequencing library totally needs 6 main enzymes, 4 enzyme reaction
systems, and cleaning and purifying for four times; therefore, it
is high in cost and complex in operation, and requires more in
operating capacity of the molecular biology for experimenter, and
it is difficult to process multiple samples synchronously.
SUMMARY OF THE APPLICATION
[0009] In view of the above problems existing in the constructing
process of the clinical plasma DNA sequencing library, the inventor
invents a new simpler and faster method for constructing the
clinical plasma DNA sequencing library, which is suitable for
various second-generation sequencing platforms, including but not
limited to the sequencing platforms such as Roche/454 FLX,
Illumina/Solexa Genome Analyzer, Applied Biosystems SOLID system,
Life Technologies Ion Torrent and the like.
[0010] The disclosure is based on the following facts: the inventor
finds that the terminus of DNA in plasma includes monophosphate
group, the 5'-terminus phosphorylation referred in the end-filling
step of the existing experimental flow is not necessary, thus T4
Polynucleotide Kinase (T4 PNK) and Adenosine Triphosphate (ATP)
need not to be used. Based on the findings above, the inventor also
finds that without using T4 PNK and ATP, the three-step reactions
of end-filling, dA-overhang and sequencing linker ligation in the
existing experimental flow cannot necessarily be performed in three
reaction systems factually, depending on the selection of enzyme
adopted for dA-overhang, but can be performed only in two reaction
systems or even only in one independent reaction system without
purification steps. Further, the inventor also finds that the DNA
library which satisfies the high-throughput sequencing requirements
also can be obtained by using a single-strand sequencing linker
instead of the double-strand sequencing linker, directly ligating
the linker to a extracted plasma DNA, and then performing
purification, PCR amplification and the like, without end-filling
and dA-overhang.
[0011] Based on the above findings, according to one aspect, the
disclosure provides a method for constructing a plasma DNA
sequencing library; which includes the steps as follows: extracting
a plasma DNA; making the plasma DNA ligate to a sequencing linker,
and purifying a ligation product; performing PCR amplification for
a purified ligation product, purifying a PCR amplification product,
and obtaining the plasma DNA sequencing library; wherein, one
important characteristic of the disclosure is that the method does
not include the step of performing 5'-terminus phosphorylation for
the plasma DNA.
[0012] Preferably, according to the method of the disclosure, the
ligation of the plasma DNA and the sequencing linker is performed
by adopting one or more ligases selected from the group consisting
of: a T4 DNA ligase, a T4 RNA ligase and a T7 DNA ligase.
[0013] According to the method of the disclosure, in some
embodiments, the sequencing linker is a single-strand sequencing
linker; and in other embodiments, the sequencing linker is a
double-strand sequencing linker.
[0014] In the case that the sequencing linker is the double-strand
sequencing linker, in one embodiment, the method of the disclosure
further includes the step of: after extracting the plasma DNA, and
before ligating the plasma DNA to the sequencing linker, performing
dA-overhang for the extracted plasma DNA, and purifying the plasma
DNA with dA-overhang. Preferably, the dA-overhang adopts klenow
ex-enzyme, or Taq enzyme, or the combination of klenow ex-enzyme
and Taq enzyme. Alternatively, the dA-overhang and the ligation of
the sequencing linker can be performed in one reaction system,
namely, the purification can be omitted after performing
dA-overhang, and the ligation of the sequencing linker is directly
performed, wherein the dA-overhang adopts the Taq enzyme.
[0015] In the case that the sequencing linker is the double-strand
sequencing linker, in another embodiment, the method of the
disclosure further includes the steps of: after extracting the
plasma DNA, and before ligating the plasma DNA to the sequencing
linker, performing end-filling and dA-overhang for an extracted
plasma DNA. Wherein, the end-filling and dA-overhang may be
performed in two reaction systems, namely, the step of dA-overhang
is performed after the step of the end-filling and the
purification; and preferably, the end-filling and dA-overhang are
performed in one reaction system, the plasma DNA is purified after
performing the end-filling and dA-overhang, wherein, the T4 DNA
polymerase is adopted in the step of end-filling , and the Taq
enzyme is adopted in the step of dA-overhang; or more preferably,
the end-filling and the dA-overhang, and the ligation between the
plasma DNA and the sequencing linker are performed in one reaction
system, wherein, the T4 DNA polymerase is adopted in the step of
the end-filling ,and the Taq enzyme is adopted in the step of
dA-overhang.
[0016] according to another aspect, the disclosure provides a kit
for constructing a plasma DNA sequencing library; the kit includes:
a reagent which ligates a plasma DNA to a sequencing linker,
including the sequencing linker, a ligase and a ligation buffer;
and reagents and instruments used for purifying a ligation product;
a reagent which performs PCR amplification for a purified ligation
product, and reagents and instruments for purifying the PCR
amplification product; wherein, one important characteristic of the
kit of the disclosure is that the kit does not include the reagent
which performs 5'-terminus phosphorylation for the plasma DNA.
[0017] Preferably, according to the kit of the disclosure, the
ligase is selected from the group consisting of: T4 DNA ligase, T4
RNA ligase and T7 DNA ligase.
[0018] According to the kit of the disclosure, in some embodiments,
the sequencing linker is a single-strand sequencing linker; and in
other embodiments, the sequencing linker is a double-strand
sequencing linker.
[0019] In the case that the sequencing linker is the double-strand
sequencing linker, in one embodiment, the kit of the disclosure
further includes: a reagent which performs dA-overhang for the
extracted plasma DNA, including Deoxyadenosine Triphosphate (dATP),
an enzyme for dA-overhang, a buffer for dA-overhang; and reagents
and instruments used for purifying the plasma DNA with dA-overhang.
Preferably, the enzyme used for dA-overhang is klenow ex-enzyme, or
Taq enzyme, or the combination of klenow ex-enzyme and Taq
enzyme.
[0020] In the case that the sequencing linker is the double-strand
sequencing linker, in another embodiment, the kit of the disclosure
further includes: a regent which respectively performs end-filling
and dA-overhang for the extracted plasma DNA, including an enzyme
for end-filling, Diethyl-Nitrophenyl Thiophosphate (dNTP), a buffer
for end-filling, Deoxyadenosine Triphosphate (dATP), an enzyme for
dA-overhang, and a buffer for dA-overhang; and the reagents and
instruments used for purifying an end-filled and dA-overhanged
plasma DNA.
[0021] In the case that the sequencing linker is the double-strand
sequencing linker, in another embodiment, the kit of the disclosure
further includes: a regent which performs end-filling and
dA-overhang for the extracted plasma DNA, including an enzyme for
end-filling, dNTP, an buffer for end-filling, dATP, an enzyme for
dA-overhang, and a buffer for dA-overhang; and the reagents and
instruments used for purifying an end-filled and dA-overhanged
plasma DNA; wherein, the end-filling and dA-overhang are performed
in one reaction system, and the T4 DNA polymerase is adopted for
end-filling; and the Taq enzyme is adopted for dA-overhang.
[0022] In the case that the sequencing linker is the double-strand
sequencing linker, in another embodiment, the kit of the disclosure
further includes: a regent which performs end-filling and
dA-overhang for the extracted plasma DNA, including an enzyme for
end-filling, dNTP, a buffer for end-filling, dATP, an enzyme for
dA-overhang, and a buffer for dA-overhang; wherein, the end-filling
and dA-overhang, and the ligation between the plasma DNA and the
sequencing linker are performed in one reaction system; the T4 DNA
polymerase is adopted for end-filling; and the Taq enzyme is
adopted for dA-overhang.
[0023] Based on the unexpected discovery that the terminus of DNA
in plasma includes a monophosphate group, the disclosure omits the
5'-terminus phosphorylation which is necessary in the existing
construction method of the plasma DNA sequencing library, and does
not use the T4 PNK and the ATP; furthermore, on this basis, the
disclosure performs the step of dA-overhang by using the normal Taq
polymerase to replace the klenow ex-enzyme, thus the buffers of
various reactions can be compatible; the steps of end-filling,
dA-overhang and linker ligation can be performed in two or only one
reaction system; additionally, the disclosure adopts the
double-strand sequencing linker instead of the single-strand
sequencing linker to perform the ligation of the linker without the
end-filling and dA-overhang steps in the prior art. Thus, the
disclosure greatly simplifies the construction of the plasma DNA
sequencing library and the experimental procedures, makes the cost
of construction of the plasma sample library more lower, the
efficiency more higher and the speed more faster, so it is
convenient for large-scale application.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 shows a method for constructing a second-generation
high-throughput plasma DNA sequencing library which is normally
adopted in the prior art.
[0025] FIG. 2 shows a method for constructing a second-generation
high-throughput plasma DNA sequencing library according to one
embodiment of the disclosure.
[0026] FIG. 3a and FIG. 3b show the electrophoresis results of the
plasma DNA sequencing library which is constructed according to the
method of the disclosure. FIG. 3a shows the electrophoresis results
of the plasma DNA sequencing library which is constructed according
to embodiments 1-4, wherein, the electrophoresis path 1 is the
plasma DNA sequencing library which is constructed as a comparison
according to the existing method; and the electrophoresis paths 2-5
are respectively the plasma DNA sequencing libraries which are
constructed according to the embodiments 1-4.
[0027] FIG. 3b shows the electrophoresis results of the plasma DNA
sequencing library which is constructed according to the embodiment
5, wherein, the electrophoresis paths 1-5 are respectively the
plasma sequencing libraries which are constructed according to the
embodiments 1-5.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0028] As mentioned above, the construction of the plasma DNA
library suitable for the second-generation high-throughput
sequencing platform at present mainly includes the steps as follows
(referring to FIG. 1): extracting a plasma DNA (Step
101)-performing end-repairing (filling), 5'-terminus
phosphorylation and purification for a extracted plasma DNA (Step
102)--performing 3' dA-overhang and purification for the 5'
phosphorylated blunt-ended DNA fragments (Step 103)-ligating the
DNA with 3' dA-overhang to the sequencing linker (Step
104)-purifying a ligation product in order to remove the unligated
linker (Step 105)-performing PCR for a purified product (Step
106)--purifying a PCR product, and obtaining the plasma DNA
sequencing library (Step 107). Wherein, the purification step needs
to be performed almost in each of the steps. Therefore, the
construction method of the second-generation plasma DNA sequencing
library totally requires 6 main enzymes, 4 enzyme reaction systems,
and cleaning and purifying for 4 times; thus this method has higher
cost and complicated operation, requires higher operating
capability for experimenter in molecular biology, and it is hard to
process multiple samples synchronously.
[0029] Via repeated experiments, the inventor unexpectedly finds
that the terminus of DNA in the plasma includes a monophosphate
group, and the 5'-terminus phosphorylation referred in the
end-filling step of the existing experimental flow is unnecessary,
thus the T4 PNK and ATP can be omitted in the construction of the
plasma DNA sequencing library; therefore, one of the important
characteristics of constructing the plasma DNA sequencing library
in the disclosure is excluding the step of 5'-terminus
phosphorylation.
[0030] On this basis, the inventor also finds that, under the
condition of not using the T4 PNK and the ATP, the end-filling,
dA-overhang and the ligation of sequencing linker in the existing
experimental flow cannot necessarily be performed in three reaction
systems, by depending on the selection of enzyme adopted in
dA-overhang, but can be performed only in two reaction systems, or
even only in one independent reaction system, without purification
step there-among.
[0031] In addition, the inventor also finds that the steps of
end-filling and dA-overhang can be omitted by adopting a
single-strand sequencing linker instead of the double-strand
sequencing linker; the DNA library which satisfies the
high-throughput sequencing requirements also can be obtained by
directly ligating the extracted plasma DNA to the linker, and then
performing the purification and PCR amplification steps and the
like.
[0032] FIG. 2 shows a method for constructing a second-generation
high-throughput plasma DNA sequencing library according to one
embodiment of the disclosure, mainly including the steps of:
extracting the plasma DNA (Step 201)--ligating the DNA to the
sequencing linker (Step 204)-purifying a ligation product in order
to remove the unligated linker (Step 205)--performing PCR for a
purified product (Step 206)--purifying a PCR product, and obtaining
a plasma DNA sequencing library (Step 207). According to another
embodiment of the disclosure, between the Step 201 and the Step
204, the method further includes: Step 202--performing
end-repairing and 5'-terminus phosphorylation for a extracted
plasma DNA, and Step 203--performing 3' dA-overhang for the 5'
phosphorylated blunt-ended DNA fragments (not shown in the
figures); the Step 202 and Step 203 can be performed in the same
reaction system; or the Step 202, Step 203 and Step 204 can be
performed in the same reaction system.
[0033] Compared with the method for constructing the plasma DNA
sequencing library in the prior art, the method of the disclosure
obviously simplifies the construction flow of the plasma DNA
sequencing library, simplifies the experimental procedures, makes
the construction of the plasma sample library have lower cost,
higher efficiency and faster speed, and is convenient for
large-scale application.
[0034] The disclosure is described as below with reference to
drawings and in conjunction with embodiments in details. It should
be noted that, the drawings and embodiments in the disclosure are
only used for the exemplification purposes for those skilled in the
art, not forming any limitation to the disclosure. The embodiments
in the application and the characteristics in the embodiments can
be mutually combined without contradiction.
Embodiment: A Method of Constructing Plasma DNA Sequencing Library
that Suitable for Next-Generation High-Throughput Sequencing
According to the Disclosure
Embodiment 1
[0035] The method for constructing a plasma DNA sequencing library
in the embodiment 1 mainly includes the steps as follows: [0036]
(1) extracting plasma DNA: this step can be performed by using any
method and reagent suitable for extracting the plasma DNA which are
known by those skilled in the art. [0037] (2) performing
end-filling, and then cleaning and purifying the products: this
step can be performed by using any method and reagent suitable for
end-filling and the following step of cleaning and purification
which are known by those skilled in the art. For example, the T4
DNA polymerase, and the Klenow can be used as the enzyme for
end-filling. [0038] (3) performing dA-overhang, and then cleaning
and purifying the products: this step can be performed by using any
method and reagent suitable for performing dA-overhang and then
cleaning and purifying the products which are known by those
skilled in the art. For example, a base can be over-hanged from the
double-strand ends by the product of the former step under the
effect of klenow ex-(New England Biolabs) (which is an improved
klenow enzyme, of which 3'-5' exonuclease is inactivity). [0039]
(4) ligating the sequencing linker, and then cleaning and purifying
the products: this step can be performed by using any method and
reagent suitable for ligating the linker and then cleaning and
purifying the products which are known by those skilled in the art.
For example, the dA-overhang and the double-strand sequencing
linker can be connected under the effect of T4 DNA ligase, T4 RNA
ligase, T7 DNA ligase, or the combination of two or more thereof.
Wherein, the sequencing linker can be commercially available or
self-synthesized. [0040] (5) performing PCR amplification for the
cleaning products obtained in the former step, and then cleaning
and purifying the products: as the PCR reaction is very mature in
this field, the selection of the primers and polymerases of the PCR
reaction, and the design of the PCR reaction circulation are also
within the capacities of those skilled in the art.
[0041] In addition, before the high-throughput sequencing, the
following operations need to be performed for the library above:
[0042] (6) using quantitative tools, such as nano drop (nano drop
technology Co., Ltd.), Bioanalyser 2100 (Agilent), Qubit (Life
Technologies) or qPCR to quantify the sequencing library; [0043]
(7) mixing an equal amount of different samples according to the
arrangement of channels of equipment and the difference of the tag
sequence; [0044] (8) samples processed by equipment.
One Embodiment for Sequencing the Plasma DNA Sample According to
the Method of the Embodiment 1 of the Disclosure is Shown below
[0045] Step 1: extracting approximate 5 ng plasma DNA.
[0046] Step 2: preparing the end-filling reaction mixture as shown
in Table 1, and incubating at 20 degrees centigrade for 30 min;
purifying the DNA samples by a purification column, and eluting the
samples with 42 .mu.l of sterile dH.sub.2O or elution buffer.
Wherein, the elution buffer adopted in the disclosure is 10 mM
Tris-CI, pH 8.0, however, the elution buffer suitable for the
disclosure is not limited thereby.
TABLE-US-00001 TABLE 1 Plasma DNA solution 40.5 .mu.l T4 DNA
polymerase buffer (10.times.) 5 .mu.l 10 mM dNTP mixture 2 .mu.l T4
DNA polymerase 2 .mu.l Klenow enzyme 0.5 .mu.l Sterile H.sub.2O 0
.mu.l Total volume 50 .mu.l
[0047] Step 3: preparing the reaction mixture which is used for
overhanging polyadenylation tail at the 3'-terminus as shown in
Table 2, and incubating at 37 degrees centigrade for 30 min;
purifying the DNA samples by a purification column, and eluting the
samples with 25 .mu.l of sterile dH.sub.2O or elution buffer.
TABLE-US-00002 TABLE 2 Blunt-ended DNA 32 .mu.l Klenow reaction
buffer (10.times.) 5 .mu.l dATP solution 10 .mu.l klenow ex-(3'-5'
exonuclease activity deletion) 3 .mu.l Sterile H.sub.2O 0 .mu.l
Total volume 50 .mu.l
[0048] Step 4: preparing the reaction mixture for ligating the DNA
fragment to the sequencing linker as shown in Table 3, and
incubating at 20 degrees centigrade for 15 min; purifying and
recycling the DNA samples on a Qiagen column, and eluting the
samples with 25 .mu.l of sterile dH.sub.2O or elution buffer.
TABLE-US-00003 TABLE 3 End-filled, dA-tail DNA 33 .mu.l Fast
ligation reaction buffer (5.times.) 10 .mu.l 5 .mu.M DNA linker 2
.mu.l Fast T4 DNA ligase (NEB) 5 .mu.l Total volume 50 .mu.l
[0049] Step 5: preparing the PCR reaction mixture as shown in Table
4, enriching the linker-modified DNA fragments by PCR; extracting
the DNA samples by a Qiagen column, and eluting the samples with 25
.mu.l of sterile dH.sub.2O or elution buffer.
TABLE-US-00004 TABLE 4 DNA 12.5 .mu.l Phusion DNA polymerase
(Phusion DNA polymerase mixture) 25 .mu.l PCR primer mixture 2
.mu.l Ultrapure water 10.5 .mu.l Total volume 50 .mu.l
[0050] The PCR reaction program is: a. 98 degrees centigrade for 30
s-b.18 circulations as below: 98 degrees centigrade for 10 s, 65
degrees centigrade for 30 s, 72 degrees centigrade for 30 s-c.72
degrees centigrade for 5 min-d. Maintaining at 4 degrees
centigrade.
[0051] Step 6: quantifying and mixing the libraries: taking 1 .mu.l
of library to quantify by Qubit; mixing an equal amount of the
sample libraries with different linkers in the same channel
according to the channel arrangement; and taking 1 .mu.l of library
to quantify by Qubit again.
[0052] Step 7: sequencing by equipment: diluting the first-end
sequencing primer, the tag primer and the second-end sequencing
primer to 100 .mu.M; operating the equipment according to the
Illumina GAIIx operating instructions. Alternatively, the
double-end sequencing can be performed, and the single-end
sequencing also can be performed, the second-end sequencing primer
can be omitted when performing the single-end sequencing.
[0053] Results: the result of sequencing by equipment shows that
the plasma DNA sequencing library constructed according to the
embodiment 1 of the disclosure can satisfy the design requirements
of the second-generation high-throughput sequencing. By
electrophoresis experiments, it finds that the constructed
sequencing library has similar bands (as shown in FIG. 3a and the
electrophoresis path 2) to the DNA library constructed by a
traditional method.
Embodiment 2
[0054] The method for constructing a plasma DNA sequencing library
of the embodiment 2 is basically similar to the embodiment 1; the
difference is that, the embodiment 2 does not include the
end-filling step, and directly performs dA-overhang and
purification for the extracted plasma DNA.
One Embodiment of Sequencing the Plasma DNA Samples by using the
Method According to the Embodiment 2 of the Disclosure is Shown as
below
[0055] Step 1: extracting approximate 5 ng of the plasma DNA.
[0056] Step 2: preparing the reaction mixture as shown in Table 5,
and incubating at 37 degrees centigrade for 30 min, so as to
overhang polyadenylation tail at the 3'-termiaus of the DNA
fragment; purifying the DNA samples by a purification column, and
eluting the samples with 25 .mu.l of sterile dH.sub.2O or elution
buffer.
TABLE-US-00005 TABLE 5 Blunt-ended DNA 32 .mu.l Klenow reaction
buffer (10.times.) 5 .mu.l klenow ex-(3'-5' exonuclease inactivity)
3 .mu.l dATP solution 10 .mu.l Sterile H.sub.2O 0 .mu.l Total
volume 50 .mu.l
[0057] Step 3: preparing the reaction mixture as shown in Table 6,
and incubating at 20 degrees centigrade for 15 min; ligating a
double-strand linker for the DNA fragment; purifying the DNA
samples by a Qiagen column, and eluting the samples with 25 .mu.l
of sterile dH.sub.2O or elution buffer.
TABLE-US-00006 TABLE 6 Blunt-ended, dA-tail DNA 33 .mu.l Fast
ligation reaction buffer (5.times.) 10 .mu.l 5 .mu.M DNA linker 2
.mu.l Fast T4 DNA ligase (NEB) 5 .mu.l Total volume 50 .mu.l
[0058] Step 4: preparing the reaction mixture as shown in Table 7,
and enriching the linker-modified DNA fragments by PCR; extracting
the DNA samples by a Qiagen column, and eluting the samples with 25
.mu.l of sterile dH.sub.2O or elution buffer.
TABLE-US-00007 TABLE 7 DNA 12.5 .mu.l Phusion DNA polymerase
(Phusion DNA polymerase mixture) 25 .mu.l PCR primer mixture 2
.mu.l Ultrapure water 10.5 .mu.l Total volume 50 .mu.l
[0059] The PCR reaction program is: a. 98 degrees centigrade for 30
s-b. 18 circulations as below: 98 degrees centigrade for 10 s, 65
degrees centigrade for 30 s, 72 degrees centigrade for 30 s-c. 72
degrees centigrade for 5 min-d. Maintaining at 4 degrees
centigrade.
[0060] Step 5: quantifying and mixing the libraries: taking 1 .mu.l
of library to quantify by Qubit; mixing an equal amount of the
sample libraries with different linkers in the same channel
according to the channel arrangement; and taking 1 .mu.l of library
to quantify by Qubit again.
[0061] Step 6: sequencing by equipment: diluting the first-end
sequencing primer, the tag primer and the second-end sequencing
primer to 100 .mu.M; operating the equipment according to the
Illumina GAIIx operating instructions. Alternatively, the
double-end sequencing can be performed, and the single-end
sequencing also can be performed, the second-end sequencing primer
can be omitted when performing the single-end sequencing.
[0062] Results: the result of sequencing by equipment shows that
the plasma DNA sequencing library constructed according to the
embodiment 2 of the disclosure can satisfy the design requirements
of the second-generation high-throughput sequencing. By
electrophoresis experiments, it finds that the constructed
sequencing library has similar bands (as shown in FIG. 3a, the
electrophoresis path 3) to the DNA library constructed by a
traditional method.
Embodiment 3
[0063] The method for constructing a plasma DNA sequencing library
of the embodiment 3 is basically similar to the embodiment 1; and
the difference is that, in the embodiment 3, the end-filling and
the dA-overhang are performed in one reaction system, the step of
cleaning and purifying is omitted between the end-filling and the
dA-overhang, wherein the dA-overhang uses the ordinary Taq enzyme
instead of the commonly-used Klenow ex-enzyme, in order to make the
buffer systems of the two reactions be compatible.
One Embodiment of Sequencing the Plasma DNA Samples by using the
Method According to the Embodiment 3 of the Disclosure is Shown as
Below
[0064] Step 1: extracting approximate 5 ng of the plasma DNA.
[0065] Step 2: preparing the reaction mixture as shown in Table 8,
and incubating at 37 degrees centigrade for 20 min (end-filling),
and then incubating at 72 degrees centigrade for 20 min
(dA-overhang), so as to perform the end-filling and dA-overhang in
one reaction system; purifying the DNA samples by a purification
column, and eluting the samples with 42 .mu.l of sterile dH.sub.2O
or elution buffer. The temperature and time of end-filling and
dA-overhang can be different according to the composition of
different reaction mixtures suitable for different design
requirements.
TABLE-US-00008 TABLE 8 Plasma DNA solution 40.5 .mu.l T4 DNA
polymerase buffer (10.times.) 5 .mu.l 10 mM dNTP mixture 2 .mu.l T4
DNA polymerase 2 .mu.l Taq polymerase 0.5 .mu.l Sterile H.sub.2O 0
.mu.l Total volume 50 .mu.l
[0066] Step 3: preparing the reaction mixture as shown in Table 9,
and incubating at 20 degrees centigrade for 15 min; ligating linker
for the DNA fragments; purifying and recycling the DNA samples by a
Qiagen column, and eluting the samples with 25 .mu.l of sterile
dH.sub.2O or elution buffer.
TABLE-US-00009 TABLE 9 End-filled, dA-tail DNA 33 .mu.l Fast
ligation reaction buffer (5.times.) 10 .mu.l 5 .mu.M DNA linker 2
.mu.l Fast T4 DNA ligase (NEB) 5 .mu.l Total volume 50 .mu.l
[0067] Step 4: preparing the reaction mixture as shown in Table 10,
enriching the linker-modified DNA fragments by PCR, extracting the
DNA samples by a Qiagen column, and eluting the samples with 25
.mu.l of sterile dH.sub.2O or elution buffer.
TABLE-US-00010 TABLE 10 DNA 12.5 .mu.l Phusion DNA polymerase
(Phusion DNA polymerase mixture) 25 .mu.l PCR primer mixture 2
.mu.l Ultrapure water 10.5 .mu.l Total volume 50 .mu.l
[0068] The PCR reaction program is: a. 98 degrees centigrade for 30
s-b. 18 circulations as below: 98 degrees centigrade for 10 s, 65
degrees centigrade for 30 s, 72 degrees centigrade for 30 s-c. 72
degrees centigrade for 5 min-d. Maintaining at 4 degrees
centigrade.
[0069] Step 5: quantifying and mixing the libraries: taking 1 .mu.l
of library to quantify by Qubit; an equal amount of mixing the
sample libraries with different linkers in the same channel
according to the channel arrangement; and taking 1 .mu.l of library
to quantify by Qubit again.
[0070] Step 6: sequencing by equipment: diluting the first-end
sequencing primer, the tag primer and the second-end sequencing
primer to 100 .mu.M; operating the equipment according to the
Illumina GAIIx operating instructions. Alternatively, the
double-end sequencing can be performed, and the single-end
sequencing also can be performed, the second-end sequencing primer
is saved when performing the single-end sequencing.
[0071] Results: the result of sequencing verification shows that
the plasma DNA sequencing library constructed according to the
embodiment 3 of the disclosure can satisfy the design requirements
of the second-generation high-throughput sequencing. By
electrophoresis experiments, it finds that the constructed
sequencing library has similar bands (as shown in FIG. 3a, the
electrophoresis path 4) to the DNA library constructed by a
traditional method.
Embodiment 4
[0072] The method for constructing a plasma DNA sequencing library
of the embodiment 4 is basically similar to the embodiment 1, and
the difference is that, in the embodiment 4, the end-filling, the
dA-overhang and the ligation of the sequencing linker are performed
in one reaction system, the step of cleaning and purifying is
omitted between the end-filling and the dA-overhang, and between
the dA-overhang and the ligation of the sequencing linker, wherein
the dA-overhang uses the ordinary Taq enzyme instead of the
commonly-used Klenow ex-enzyme, in order to make the buffer systems
of the three reactions be compatible.
One Embodiment of Sequencing the Plasma DNA Samples by using the
Method According to the Embodiment 4 of the Disclosure is Shown as
Below
[0073] Step 1: extracting approximate 5 ng of the plasma DNA.
[0074] Step 2: preparing the reaction mixture as shown in Table 11,
incubating at 37 degrees centigrade for 20 min (end-filling), and
then incubating at 72 degrees for 20 min (dA-overhang), so as to
perform the end-filling and dA-overhang; the purification step is
not performed here.
TABLE-US-00011 TABLE 11 Plasma DNA solution 40.5 .mu.l T4 DNA
polymerase buffer (10.times.) 5 .mu.l 10 mM dNTP mixture 2 .mu.l T4
DNA polmyerase 2 .mu.l Taq polymerase 0.5 .mu.l Sterile H.sub.2O 0
.mu.l Total volume 50 .mu.l
[0075] Step 3: adding extra reaction agent into the former reaction
solution to prepare the reaction mixture as shown in Table 12,
incubating at 20 degrees centigrade for 15 min, in order to ligate
sequencing linker to the DNA fragments; purifying and recycling the
DNA samples by a Qiagen column, and eluting the samples with 25
.mu.l of sterile dH.sub.2O or elution buffer.
TABLE-US-00012 TABLE 12 Reaction solution of the former step 50
.mu.l Fast ligation reaction buffer (10.times.) 10 .mu.l 5 .mu.M
DNA linker 2 .mu.l Fast T4 DNA ligase (NEB) 5 .mu.l Total volume
100 .mu.l
[0076] Step 4: preparing the reaction mixture as shown in Table 13,
enriching the linker-modified DNA fragments by PCR, extracting the
DNA samples by a Qiagen column, and eluting the samples with 25
.mu.l of sterile dH.sub.2O or elution buffer.
TABLE-US-00013 TABLE 13 DNA 12.5 .mu.l Phusion DNA polymerase
(Phusion DNA polymerase mixture) 25 .mu.l PCR primer mixture 2
.mu.l Ultrapure water 10.5 .mu.l Total volume 50 .mu.l
[0077] The PCR reaction program is: a. 98 degrees centigrade for 30
s-b. 18 circulations as below: 98 degrees centigrade for 10 s, 65
degrees centigrade for 30 s, 72 degrees centigrade for 30 s-c. 72
degrees centigrade for 5 min-d. Maintaining at 4 degrees
centigrade.
[0078] Step 5: quantifying and mixing the libraries: taking 1 .mu.l
of library to quantify by Qubit; an equal amount of mixing the
sample libraries with different linkers in the same channel
according to the channel arrangement; and taking 1 .mu.l of library
to quantify by Qubit again.
[0079] Step 6: sequencing by equipment: diluting the first-end
sequencing primer, the tag primer and the second-end sequencing
primer to 100 .mu.M; operating the equipment according to the
Illumina GAIIx operating instructions. Alternatively, the
double-end sequencing can be performed, and the single-end
sequencing also can be performed, the second-end sequencing primer
is saved when performing the single-end sequencing.
[0080] Results: the result of sequencing by equipment shows that
the plasma DNA sequencing library constructed according to the
embodiment 3 of the disclosure can satisfy the design requirements
of the second-generation high-throughput sequencing. By
electrophoresis experiments, it finds that the constructed
sequencing library has similar bands (as shown in FIG. 3a, the
electrophoresis path 5) to the DNA library constructed by a
traditional method.
Embodiment 5
[0081] The method for constructing a plasma DNA sequencing library
of the embodiment 5 is basically similar to the embodiment 1, and
the difference is that, in the embodiment 5, the steps of
end-filling and the dA-overhang are omitted; the extracted plasma
DNA is directly ligated to the sequencing linker, and the
single-strand sequencing linker is adopted to replace the
double-strand sequencing linker in embodiment 1. The inventor finds
that the plasma DNA library which satisfies the high-throughput
sequencing requirements also can be obtained by adopting the
single-strand sequencing liker instead of the double-strand
sequencing linker, without performing the steps of end-filling and
dA-overhang.
The Embodiments 1-5 of Sequencing the Plasma DNA Samples by using
the Method According to the Embodiment 5 of the Disclosure are
Shown as Below
[0082] Step 1: extracting approximate 5 ng of the plasma DNA.
[0083] Step 2: respectively preparing the reaction mixture as shown
in Table 14, incubating at 20 degrees centigrade for 60 min, so as
to ligate linker to the DNA fragment; purifying and recycling the
DNA samples by a Qiagen column and eluting the samples with 25
.mu.l of sterile dH.sub.2O or elution buffer. Alternatively, the T4
DNA ligase and the T4 RNA enzyme can have the proportional
relations different from each specific embodiment.
TABLE-US-00014 TABLE 14 Reagent Embodiment 1 Embodiment 2
Embodiment 3 Embodiment 4 Embodiment 5 Plasma DNA 42 .mu.l 39 .mu.l
37 .mu.l 37 .mu.l 42 .mu.l solution Fast ligation 5 .mu.l 5 .mu.l 5
.mu.l 5 .mu.l 5 .mu.l reaction buffer (10.times.) 5 .mu.M 2 .mu.l 2
.mu.l 2 .mu.l 2 .mu.l 2 .mu.l single-strand DNA linker T4 DNA 0
.mu.l 2 .mu.l 4 .mu.l 5 .mu.l 1 .mu.l ligase (NEB) T4 RNA 1 .mu.l 2
.mu.l 2 .mu.l 1 .mu.l 0 .mu.l ligase (NEB) Total volume 50 .mu.l 50
.mu.l 50 .mu.l 50 .mu.l 50 .mu.l
[0084] Step 3: preparing the PCR reaction mixture as shown in Table
15, enriching the linker-modified DNA fragments by PCR; extracting
the DNA samples by a Qiagen column, and eluting the samples with 25
.mu.l of sterile dH.sub.2O or elution buffer.
TABLE-US-00015 TABLE 15 DNA 23 .mu.l Phusion DNA polymerase
(Phusion DNA polymerase mixture) 25 .mu.l PCR primer mixture 2
.mu.l Ultrapure water 0 .mu.l Total volume 50 .mu.l
[0085] Because of adopting the single-strand sequencing linker, the
PCR reaction program is adjusted as follows: 72 degrees centigrade
for 5 min-98degrees centigrade for 30 s-4 circulations as follows:
98 degrees centigrade for 10 s, 30 degrees centigrade for 30 s, 72
degrees centigrade for 30 s-d. 98 degrees centigrade for 30 s-e. 16
circulations as follows: 98 degrees centigrade for 10 s, 65 degrees
centigrade for 30 s, 72 degrees centigrade for 30 s-f. 72 degrees
centigrade for 5 min-g. Maintaining at 4 degrees centigrade.
[0086] Step 4: quantifying and mixing the libraries: taking 1 .mu.l
of library to quantify by Qubit; an equal amount of mixing the
sample libraries with different linkers in the same channel
according to the channel arrangement; and taking 1 .mu.l of library
to quantify by Qubit again.
[0087] Step 5: sequencing by equipment: diluting the first-end
sequencing primer, the tag primer and the second-end sequencing
primer to 100 .mu.M; operating the equipment according to the
Illumina GAIIx operating instructions. Alternatively, the
double-end sequencing can be performed, and the single-end
sequencing also can be performed, the second-end sequencing primer
can be omitted when performing the single-end sequencing.
[0088] Results: the result of sequencing by equipment shows that
the plasma DNA sequencing library constructed according to the
embodiment 5 of the disclosure can satisfy the design requirements
of the second-generation high-throughput sequencing. By
electrophoresis experiments (as shown in FIG. 3b), it finds that
the constructed sequencing library has similar bands to the DNA
library which is constructed by a traditional method; and the
similar electrophoresis bands can be obtained by adopting different
proportions of T4 DNA ligase and T4 RMA enzymes.
[0089] In addition, the disclosure also provides a kit for
constructing a DNA plasma sequencing library according to the
embodiments 1-5 of the disclosure. According to different
embodiments, the kit can include the enzyme for filling and
repairing the plasma DNA end, the enzyme for overhanging adenine at
the 3'-terminus of the DNA, including T4 DNA polymerase, Klenow
enzyme, DNA polymerase, Taq enzyme and Klenow ex-(3'-5' exonuclease
inactivity deletion); the enzyme for ligating the linker, including
T4 DNA ligase, T4 RNA ligase, T7 DNA ligase, or the combinations
thereof; the polymerase for performing the PCR amplification,
including Phusion DNA polymerase and the like; and the buffers used
for various enzymes; and the dNTP required by end-filling and PCR
amplification, dATP required by dA-overhang, the double-strand
sequencing linker or single-strand sequencing linker for the linker
ligation, primers for the PCR amplification and the like.
[0090] As the disclosure finds that the 5'-terminus phosphorylation
step can be omitted during the construction of a plasma DNA
sequencing library, one important characteristic of the kit of the
disclosure is that the reagent for performing 5'-terminus
phosphorylation for the plasma DNA can be excluded.
[0091] In addition, as the disclosure adopts the Taq enzyme to
perform dA-overhang without performing the 5'-terminus
phosphorylation, the end-filling and dA-overhang can be performed
in one reaction system, or the end-filling, dA-overhang and the
ligation of the sequencing linker can be performed in one reaction
system; thus, the kit of the disclosure may exclude the instruments
and reagents for purifying after performing the end-filling, and/or
the instruments and reagents for purifying after performing
dA-overhang, consequently reducing the library construction cost,
and simplifying the library construction flows.
[0092] If necessary, the kit of the disclosure further includes
reagents and instruments for purifying end-filling products,
reagents and instruments for purifying dA-overhang products,
reagents and instruments for purifying ligation products of
sequencing linker; and reagents and instruments for purifying PCR
products and the like. The purification reagents can include the
sterile dH.sub.2O or elution buffer; purification devices can
include purification columns or Qiagen columns. The purification
reagent and instruments adopted in the disclosure are not limited
by the specific reagents and instruments listed in this text, each
of the purification reagents and instruments which are commonly
used in the field can be applied in this disclosure according to
the judgment of the technical staff.
[0093] It should be noted that, the above is only the preferred
embodiment of the disclosure, but not intended to limit the scope
of protection of the disclosure; for those skilled in the art,
various changes and modifications can be made to the disclosure.
For example, the dA-overhang step and the sequencing linker
ligation step above also can be performed in one system, namely,
the extracted plasma DNA can be performed with dA-overhang step,
and the linker ligation step can be directly performed without
performing the purification step. In addition, although the shown
enzyme for ligating the sequencing linker is T4 DNA polymerase
and/or T4 RNA polymerase, the T7 DNA polymerase, or the combination
of the T7 DNA polymerase and the T4 DNA polymerase, and/or the
combination of the T7 DNA polymerase and the T4 RNA polymerase also
can be independently used. The reaction buffer, the PCR reaction
flow and the like referred in the construction of the plasma DNA
sequencing library also can be adjusted and changed correspondingly
according to the specific requirements. Those skilled in the art
should understand that any modifications, equivalent replacements,
improvements and the like within the spirit and principle of the
disclosure should fall within the scope of protection of the
disclosure.
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