分子生物学张尚宏课件分子生物学
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分子生物学全套课件96P课件
Molecular Biology: The biology (structure and function) of macro-molecules
Protein Nucleic acid (DNA/ RNA/
Coding-/ non-coding/ functional/ junk/ ... Fatty acid Polysaccharide (sugar) Small molecules
Then what?
Amplification (PCR) +Separation
ROCHE Strategy
Emulsion PCR; 1 fragment/ bead
Primer
dNTP (each)
Polymerase
Template
Not clearly demonstrated
PCR amplification (Chain extension) Emulsion breaking Template dissociation
FOCUSing on NUCLEIC ACID; Molecular Genetics
DNA Sequrncing
Cost: Speed: Effeciency: Method:
Generation: DNA cloning:
...:
Expensive – morderately expensive – relatively cheap Time consumming – below endurnce – fast Low – fast - high throughput PAGE+autoradiography – automatic – massively paralell reversible chain termination + length based detection -
分子生物学全套课件96P课件
in situ amplification + fluorescene detection 1st – 2nd – 3rd (so called next generation) Have to – Cloning + PCR – PCR only (highly effecient way) ...
4 two base combinations each color for a given sequence, 1 combination is true and detectable explain the color space alignment with reference 5 colors - 25 bases – unique in genome repeats? The same as others
Sulfurylase
In one tube in one machine Automated process Light strength is absolutely proportional to ATP, thus to PPi to base on the template
ATP (+ sulfate)
template
Primer Machine + Kit
DNAn + dNTP
extension
polymerase
DNAn+1 + PPi
Over supplied
+
adenosine-5’-phosphosulfate, APS
dNTP
Apyrase
dNDP + dNMP + Pi ADP + AMP + Pi
4 two base combinations each color for a given sequence, 1 combination is true and detectable explain the color space alignment with reference 5 colors - 25 bases – unique in genome repeats? The same as others
Sulfurylase
In one tube in one machine Automated process Light strength is absolutely proportional to ATP, thus to PPi to base on the template
ATP (+ sulfate)
template
Primer Machine + Kit
DNAn + dNTP
extension
polymerase
DNAn+1 + PPi
Over supplied
+
adenosine-5’-phosphosulfate, APS
dNTP
Apyrase
dNDP + dNMP + Pi ADP + AMP + Pi
分子生物学全套课件96P课件精品文档
分子生物学
Molecular Biology
Presumed that you have learned “Molecular Biology” before your receiving the Bachelor’s Degree of Sciences;
Designed for helping you to become associated with the most researching interests (areas) of our university;
+
lucifCAAAACCCC
luciferase
Light + oxylufiferin
AC G TG
Pyrograph
Recorded by computer
dNTP added in serial
Sequencing by ligation: Applied Biosystems
polony
20 microns
Position overlapped one base one image color order = base adding order = sequence
Reversible terminators: Illumina
Bridge amplification of DNA fragments is randomly distributed across eight channels of a glass slide, to which high-density forward and reverse primers are covalently attached. The solid-phase amplification produces ~80 million molecule clusters (MCs) from individual ssDNA templates. A primer is annealed to the free ends of templates in each MC. The polymerase extends and then terminates DNA synthesis from a set of four reversible terminators (RTs), each labeled with a different dye. Unincorporated RTs are washed away, base identification is performed by four-colour imaging, and blocking and dye groups are removed by chemical cleavage to permit the next cycle. Colour images for a given MC provide reads of ~45 bases. Substitutions are the most common error type.
Molecular Biology
Presumed that you have learned “Molecular Biology” before your receiving the Bachelor’s Degree of Sciences;
Designed for helping you to become associated with the most researching interests (areas) of our university;
+
lucifCAAAACCCC
luciferase
Light + oxylufiferin
AC G TG
Pyrograph
Recorded by computer
dNTP added in serial
Sequencing by ligation: Applied Biosystems
polony
20 microns
Position overlapped one base one image color order = base adding order = sequence
Reversible terminators: Illumina
Bridge amplification of DNA fragments is randomly distributed across eight channels of a glass slide, to which high-density forward and reverse primers are covalently attached. The solid-phase amplification produces ~80 million molecule clusters (MCs) from individual ssDNA templates. A primer is annealed to the free ends of templates in each MC. The polymerase extends and then terminates DNA synthesis from a set of four reversible terminators (RTs), each labeled with a different dye. Unincorporated RTs are washed away, base identification is performed by four-colour imaging, and blocking and dye groups are removed by chemical cleavage to permit the next cycle. Colour images for a given MC provide reads of ~45 bases. Substitutions are the most common error type.
分子生物学全套课件96P课件
分子生物学
Molecular Biology
Presumed that you have learned “Molecular Biology” before your receiving the Bachelor’s Degree of Sciences;
Designed for helping you to become associated with the most researching interests (areas) of our university;
polony
20 microns
Position overlapped one base one image color order = base adding order = sequence
Reversible terminators: Illumina
Bridge amplification of DNA fragments is randomly distributed across eight channels of a glass slide, to which high-density forward and reverse primers are covalently attached. The solid-phase amplification produces ~80 million molecule clusters (MCs) from individual ssDNA templates. A primer is annealed to the free ends of templates in each MC. The polymerase extends and then terminates DNA synthesis from a set of four reversible terminators (RTs), each labeled with a different dye. Unincorporated RTs are washed away, base identification is performed by four-colour imaging, and blocking and dye groups are removed by chemical cleavage to permit the next cycle. Colour images for a given MC provide reads of ~45 bases. Substitutions are the most common error type.
Molecular Biology
Presumed that you have learned “Molecular Biology” before your receiving the Bachelor’s Degree of Sciences;
Designed for helping you to become associated with the most researching interests (areas) of our university;
polony
20 microns
Position overlapped one base one image color order = base adding order = sequence
Reversible terminators: Illumina
Bridge amplification of DNA fragments is randomly distributed across eight channels of a glass slide, to which high-density forward and reverse primers are covalently attached. The solid-phase amplification produces ~80 million molecule clusters (MCs) from individual ssDNA templates. A primer is annealed to the free ends of templates in each MC. The polymerase extends and then terminates DNA synthesis from a set of four reversible terminators (RTs), each labeled with a different dye. Unincorporated RTs are washed away, base identification is performed by four-colour imaging, and blocking and dye groups are removed by chemical cleavage to permit the next cycle. Colour images for a given MC provide reads of ~45 bases. Substitutions are the most common error type.
分子生物学全套课件96P课件
FOCUSing on NUCLEIC ACID; Molecular Genetics
DNA Sequrncing
Cost: Speed: Effeciency: Method:
Generation: DNA cloning:
...:
Expensive – morderately expensive – relatively cheap Time consumming – below endurnce – fast Low – fast - high throughput PAGE+autoradiography – automatic – massively paralell reversible chain termination + length based detection -
Then what?
Amplification (PCR) +Separation
ROCHE Strategy
Emulsion PCR; 1 fragment/ bead
Primer
dNTP (each)
Polymerase
Template
Not clearly demonstrated
PCR amplification (Chain extension) Emulsion breaking Template dissociation
Reads, not template
The recorded is the sequence of dNTP flow; FLOWGRAM; Reads averaging 400 bases; Homopolymer repeats up to six nucleotides; The number of dNTPs added is directly proportional to the light signal, with fluctuation.
DNA Sequrncing
Cost: Speed: Effeciency: Method:
Generation: DNA cloning:
...:
Expensive – morderately expensive – relatively cheap Time consumming – below endurnce – fast Low – fast - high throughput PAGE+autoradiography – automatic – massively paralell reversible chain termination + length based detection -
Then what?
Amplification (PCR) +Separation
ROCHE Strategy
Emulsion PCR; 1 fragment/ bead
Primer
dNTP (each)
Polymerase
Template
Not clearly demonstrated
PCR amplification (Chain extension) Emulsion breaking Template dissociation
Reads, not template
The recorded is the sequence of dNTP flow; FLOWGRAM; Reads averaging 400 bases; Homopolymer repeats up to six nucleotides; The number of dNTPs added is directly proportional to the light signal, with fluctuation.
分子生物学全套课件96P课件
Sulfurylase
In one tube in one machine Automated process Light strength is absolutely proportional to ATP, thus to PPi to base on the template
ATP (+ sulfate)
分子生物学
Molecular Biology
Presumed that you have learned “Molecular Biology” before your receiving the Bachelor’s Degree of Sciences;
Designed for helping you to become associated with the most researching interests (areas) of our university;
Molecular Biology: The biology (structure and function) of macro-molecules
Protein Nucleic acid (DNA/ RNA/
Coding-/ non-coding/ functional/ junk/ ... Fatty acid Polysaccharide (sugar) Small molecules
Reads, not template
The recorded is the sequence of dNTP flow; FLOWGRAM; Reads averaging 400 bases; Homopolymer repeats up to six nucleotides; The number of dNTPs added is directly proportional to the light signal, with fluctuation.
分子生物学全套课件96P课件
Then what?
Amplification (PCR) +Separation
ROCHE Strategy
Emulsion PCR; 1 fragment/ bead
Primer
dNTP (each)
Polymerase
Template
Not clearly demonstrated
PCR amplification (Chain extension) Emulsion breaking Template dissociation
分子生物学
Molecular Biology
Presumed that you have learned “Molecular Biology” before your receiving the Bachelor’s Degree of Sciences;
Designed for helping you to become associated with the most researching interests (areas) of our university;
Sulfurylase
In one tube in one machine Automated process Light strength is absolutely proportional to ATP, thus to PPi to base on the template
ATP (+ sulfate)
Reads, not template
The recorded is the sequence of dNTP flow; FLOWGRAM; Reads averaging 400 bases; Homopolymer repeats up to six nucleotides; The number of dNTPs added is directly proportional to the light signal, with fluctuation.
分子生物学全套课件96P课件
分子生物学
Molecular Biology
Presumed that you have learned “Molecular Biology” before your receiving the Bachelor’s Degree of Sciences;
Designed for helping you to become associated with the most researching interests (areas) of our university;
Emulsion PCR: 1 – 2 - 2n; synchronically,
PicoTiterPlate
adenosi3; sulphate
SCcoamnnpiuntger+
Pyrosequencing: Templates are prepared by emPCR, with 1–2 million beads deposited into PTP wells. Smaller beads with attached sulphurylase and luciferase surround the template beads. Individual dNTPs flow sequentially across the wells, dispensed in a predetermined order. On incorporation of the complement dNTP, released PPi is converted to ATP, producing light from the oxidation of luciferin to oxyluciferin. Reads averaging 400 bases are recorded as flowgrams. For homopolymer repeats up to six nucleotides, the number of dNTPs added is directly proportional to the light signal. Insertions are the most common error type, followed by deletions.
Molecular Biology
Presumed that you have learned “Molecular Biology” before your receiving the Bachelor’s Degree of Sciences;
Designed for helping you to become associated with the most researching interests (areas) of our university;
Emulsion PCR: 1 – 2 - 2n; synchronically,
PicoTiterPlate
adenosi3; sulphate
SCcoamnnpiuntger+
Pyrosequencing: Templates are prepared by emPCR, with 1–2 million beads deposited into PTP wells. Smaller beads with attached sulphurylase and luciferase surround the template beads. Individual dNTPs flow sequentially across the wells, dispensed in a predetermined order. On incorporation of the complement dNTP, released PPi is converted to ATP, producing light from the oxidation of luciferin to oxyluciferin. Reads averaging 400 bases are recorded as flowgrams. For homopolymer repeats up to six nucleotides, the number of dNTPs added is directly proportional to the light signal. Insertions are the most common error type, followed by deletions.