Molecule Techniques

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Thematic presentation:

MOLECULE TECHNIQUES

MSc. Ton Bao Linh

Nong Lam University

Department of Biotechnology


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MOLECULE

TECHNIQUES


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Members

1. Hoang Thi Huyen Trang 11126237 DH11SH

2. Nguyen Thi Hong Phuc 11126183 DH11SH

3. Le Dang Huynh Tram 11126241 DH11SH4. Le Nguyen Thao Ly 11126308 DH11SH

5. Le Nhat Tan 11126321 DH11SH


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Sections

Molecule techniques

Electrophoresis

PCR

Hybridization


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Principle

In most cases, the purpose of the

hybridization techniques is identification or

localization of certain nucleic acid

sequences in the genome of somespecies. Two basic notions are used: the

target molecule representing the DNA,

RNA or protein sequence that should be

identified and the probe molecule whoidentify the target, by hybridization.


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Categories


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Hybridization stages

1. Probe synthesis 2. Probe marking

3. Target DNAprocessing assumesthe source

4. Target DNAdenaturation

5. Target DNA transferto

6. Molecularhybridization


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Southern Blot

EdwinMellor

Southern


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The original methodology


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Stages


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Modifications

First nylon membranes are less fragile thannitrocellulose sheets

Certain conditions the transferred DNAbecomes covalently bound to the membraneduring the transfer process

Nylon membranes efficiently bind DNA fragmentsdown to 50 bp in length, where as nitrocellulosemembranes are effective only with moleculeslonger than 500 bp


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Applications and Limitations

The applications of Southern blotting are diverse andare not easily summarized in a short article. Two

examples will suffice to illustrate the range of research

questions to which the technique can be applied.

Southern blotting in cloneidentification.

Restriction fragment lengthpolymorphism analysis.


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RFLP

In molecular biology, restriction fragmentlength polymorphism is a technique that

exploits variations in homologous DNA

sequences. It refers to a difference between

samples of homologous DNA molecules thatcome from differing locations of restriction

enzyme sites, and to a related laboratory

technique by which these segments can be

illustrated. In RFLP analysis, the DNA sample isbroken into pieces (digested) by restriction

enzymes and the resulting restriction

fragments are separated according to their

lengths by gel electrophoresis.
http://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Polymorphism_(biology)http://en.wikipedia.org/wiki/Homology_(biology)http://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Restriction_sitehttp://en.wikipedia.org/wiki/Restriction_sitehttp://en.wikipedia.org/wiki/Restriction_enzymeshttp://en.wikipedia.org/wiki/Restriction_enzymeshttp://en.wikipedia.org/wiki/Gel_electrophoresishttp://en.wikipedia.org/wiki/Gel_electrophoresishttp://en.wikipedia.org/wiki/Gel_electrophoresishttp://en.wikipedia.org/wiki/Gel_electrophoresishttp://en.wikipedia.org/wiki/Restriction_enzymeshttp://en.wikipedia.org/wiki/Restriction_enzymeshttp://en.wikipedia.org/wiki/Restriction_enzymeshttp://en.wikipedia.org/wiki/Restriction_sitehttp://en.wikipedia.org/wiki/Restriction_sitehttp://en.wikipedia.org/wiki/Restriction_sitehttp://en.wikipedia.org/wiki/Restriction_sitehttp://en.wikipedia.org/wiki/Restriction_sitehttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Homology_(biology)http://en.wikipedia.org/wiki/Polymorphism_(biology)http://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Molecular_biology


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Microarray

A microarray is a multiplex lab-on-a-chip. Itis a 2D array on a solid substrate (usually

a glass slide or silicon thin-film cell)

that assays large amounts of biological

material using high-throughput

screening methods. The concept and

methodology of microarrays was first

introduced and illustrated in antibodymicroarrays (also referred to as antibody

matrix) in 1983 in a scientific publication and

a series of patents
http://en.wikipedia.org/wiki/Multiplex_(assay)http://en.wikipedia.org/wiki/Lab-on-a-chiphttp://en.wikipedia.org/wiki/Substrate_(materials_science)http://en.wikipedia.org/wiki/Glass_slidehttp://en.wikipedia.org/wiki/Silicon_thin-film_cellhttp://en.wikipedia.org/wiki/Assayshttp://en.wikipedia.org/wiki/Biotic_materialhttp://en.wikipedia.org/wiki/Biotic_materialhttp://en.wikipedia.org/wiki/High-throughput_screeninghttp://en.wikipedia.org/wiki/High-throughput_screeninghttp://en.wikipedia.org/wiki/Antibody_microarrayhttp://en.wikipedia.org/wiki/Antibody_microarrayhttp://en.wikipedia.org/wiki/Antibody_matrixhttp://en.wikipedia.org/wiki/Antibody_matrixhttp://en.wikipedia.org/wiki/Antibody_matrixhttp://en.wikipedia.org/wiki/Antibody_matrixhttp://en.wikipedia.org/wiki/Antibody_matrixhttp://en.wikipedia.org/wiki/Antibody_microarrayhttp://en.wikipedia.org/wiki/Antibody_microarrayhttp://en.wikipedia.org/wiki/Antibody_microarrayhttp://en.wikipedia.org/wiki/High-throughput_screeninghttp://en.wikipedia.org/wiki/High-throughput_screeninghttp://en.wikipedia.org/wiki/High-throughput_screeninghttp://en.wikipedia.org/wiki/High-throughput_screeninghttp://en.wikipedia.org/wiki/High-throughput_screeninghttp://en.wikipedia.org/wiki/Biotic_materialhttp://en.wikipedia.org/wiki/Biotic_materialhttp://en.wikipedia.org/wiki/Biotic_materialhttp://en.wikipedia.org/wiki/Assayshttp://en.wikipedia.org/wiki/Silicon_thin-film_cellhttp://en.wikipedia.org/wiki/Silicon_thin-film_cellhttp://en.wikipedia.org/wiki/Silicon_thin-film_cellhttp://en.wikipedia.org/wiki/Silicon_thin-film_cellhttp://en.wikipedia.org/wiki/Silicon_thin-film_cellhttp://en.wikipedia.org/wiki/Silicon_thin-film_cellhttp://en.wikipedia.org/wiki/Silicon_thin-film_cellhttp://en.wikipedia.org/wiki/Glass_slidehttp://en.wikipedia.org/wiki/Glass_slidehttp://en.wikipedia.org/wiki/Glass_slidehttp://en.wikipedia.org/wiki/Substrate_(materials_science)http://en.wikipedia.org/wiki/Substrate_(materials_science)http://en.wikipedia.org/wiki/Substrate_(materials_science)http://en.wikipedia.org/wiki/Lab-on-a-chiphttp://en.wikipedia.org/wiki/Lab-on-a-chiphttp://en.wikipedia.org/wiki/Lab-on-a-chiphttp://en.wikipedia.org/wiki/Lab-on-a-chiphttp://en.wikipedia.org/wiki/Lab-on-a-chiphttp://en.wikipedia.org/wiki/Lab-on-a-chiphttp://en.wikipedia.org/wiki/Lab-on-a-chiphttp://en.wikipedia.org/wiki/Multiplex_(assay)


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Applications

Help researchers to learn more about many

different diseases including heart disease,

mental illness and infectious diseases

Additionally, by examining the differences ingene activity between untreated and

treated tumor cells.


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The polymerase chain reaction(PCR) is

a biochemical technology in molecular

biologyto amplifya single or a few copiesof a piece of DNAacross several orders of

magnitude, generating thousands to

millions of copies of a particular DNA

sequence.
http://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/DNA_replicationhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/DNA_sequencehttp://en.wikipedia.org/wiki/DNA_sequencehttp://en.wikipedia.org/wiki/DNA_sequencehttp://en.wikipedia.org/wiki/DNA_sequencehttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/DNA_replicationhttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Molecular_biology


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Developed in 1983 by Kary Mullis

In 1993, Mullis was awarded the NobelPrize in Chemistryalong with Michael

Smithfor his work on PCR.

History
http://en.wikipedia.org/wiki/Kary_Mullishttp://en.wikipedia.org/wiki/Nobel_Prize_in_Chemistryhttp://en.wikipedia.org/wiki/Nobel_Prize_in_Chemistryhttp://en.wikipedia.org/wiki/Michael_Smith_(chemist)http://en.wikipedia.org/wiki/Michael_Smith_(chemist)http://en.wikipedia.org/wiki/Michael_Smith_(chemist)http://en.wikipedia.org/wiki/Michael_Smith_(chemist)http://en.wikipedia.org/wiki/Nobel_Prize_in_Chemistryhttp://en.wikipedia.org/wiki/Nobel_Prize_in_Chemistryhttp://en.wikipedia.org/wiki/Kary_Mullishttp://en.wikipedia.org/wiki/Kary_Mullishttp://en.wikipedia.org/wiki/Kary_Mullis


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The method relies on thermal cycling,

consist of cycles of repeated heating andcooling of the reaction for DNA melting

and enzymatic replication of the DNA


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Components

DNA templatethat contains the DNA region(target) to be amplified.

Twoprimersthat are complementarytothe 3'(three prime) ends of each of the senseand anti-sensestrand of the DNA target.

Taq polymeraseor another DNA polymerasewitha temperature optimum at around 70 C.
http://en.wikipedia.org/wiki/Primer_(molecular_biology)http://en.wikipedia.org/wiki/Complementarity_(molecular_biology)http://en.wikipedia.org/wiki/Directionality_(molecular_biology)http://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Taq_polymerasehttp://en.wikipedia.org/wiki/DNA_polymerasehttp://en.wikipedia.org/wiki/DNA_polymerasehttp://en.wikipedia.org/wiki/Taq_polymerasehttp://en.wikipedia.org/wiki/Taq_polymerasehttp://en.wikipedia.org/wiki/Taq_polymerasehttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Directionality_(molecular_biology)http://en.wikipedia.org/wiki/Complementarity_(molecular_biology)http://en.wikipedia.org/wiki/Primer_(molecular_biology)


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Deoxynucleoside triphosphates(dNTPs,sometimes called "deoxynucleotidetriphosphates)

Buffer solution, providing a suitable chemicalenvironment for optimum activity and stability ofthe DNA polymerase.

Divalentcations, magnesiumor manganeseions(Mg2+, Mn2+)

Components
http://en.wikipedia.org/wiki/Buffer_solutionhttp://en.wikipedia.org/wiki/Divalenthttp://en.wikipedia.org/wiki/Cationshttp://en.wikipedia.org/wiki/Magnesiumhttp://en.wikipedia.org/wiki/Manganesehttp://en.wikipedia.org/wiki/Manganesehttp://en.wikipedia.org/wiki/Magnesiumhttp://en.wikipedia.org/wiki/Cationshttp://en.wikipedia.org/wiki/Divalenthttp://en.wikipedia.org/wiki/Buffer_solution


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(1) Denaturing

at 9496 C.

(2) Annealingat ~65 C

(3) Elongation

at 72 C

PCR principles and proceduce


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Figure 3: Ethidium bromide-stained PCR products

after gel electrophoresis

PCR principles and proceduce
http://en.wikipedia.org/wiki/Gel_electrophoresishttp://en.wikipedia.org/wiki/File:Roland_Gel.JPGhttp://en.wikipedia.org/wiki/Gel_electrophoresishttp://en.wikipedia.org/wiki/Gel_electrophoresishttp://en.wikipedia.org/wiki/Gel_electrophoresis


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PCR stages


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Detecting infectiousagents

The role of PCR incancer diagnostics

Genetic diseases andaternit testin

Application of PCR


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Figure : Electrophoresis of PCR-amplified DNA

fragments. (1) Father. (2) Child. (3) Mother. The child

has inherited some, but not all of the fingerprint of each

of its parents, giving it a new, unique fingerprint.
http://en.wikipedia.org/wiki/File:Pcr_fingerprint.png


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Amplification and quantification of DNA:

Real-time PCRis an established tool for DNAquantification that measures the accumulation

of DNA product after each round of PCR

amplification.

Application of PCR
http://en.wikipedia.org/wiki/Real-time_polymerase_chain_reactionhttp://en.wikipedia.org/wiki/Real-time_polymerase_chain_reactionhttp://en.wikipedia.org/wiki/Real-time_polymerase_chain_reactionhttp://en.wikipedia.org/wiki/Real-time_polymerase_chain_reactionhttp://en.wikipedia.org/wiki/Real-time_polymerase_chain_reaction


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Electrophoresis

Electrophoresis is the motion of dispersedparticles relative to a fluid under the influence of a

spatially uniform electric field. This electrokinetic

phenomenonwas observed for the first time in 1807

by Ferdinand Frederic Reuss, who noticed that theapplication of a constant electric

field caused clay particles dispersed in water to

migrate. It is ultimately caused by the presence of a

charged interface between the particle surface and

the surrounding fluid. It is the basis for a number ofanalytical techniques used in biochemistry for

separating molecules by size, charge, or binding

affinity.
http://en.wikipedia.org/wiki/Interface_and_colloid_sciencehttp://en.wikipedia.org/wiki/Interface_and_colloid_sciencehttp://en.wikipedia.org/wiki/Electric_fieldhttp://en.wikipedia.org/wiki/Electrokinetic_phenomenahttp://en.wikipedia.org/wiki/Electrokinetic_phenomenahttp://en.wikipedia.org/wiki/Electric_fieldhttp://en.wikipedia.org/wiki/Electric_fieldhttp://en.wikipedia.org/wiki/Clayhttp://en.wikipedia.org/wiki/Waterhttp://en.wikipedia.org/wiki/Waterhttp://en.wikipedia.org/wiki/Clayhttp://en.wikipedia.org/wiki/Electric_fieldhttp://en.wikipedia.org/wiki/Electric_fieldhttp://en.wikipedia.org/wiki/Electric_fieldhttp://en.wikipedia.org/wiki/Electrokinetic_phenomenahttp://en.wikipedia.org/wiki/Electrokinetic_phenomenahttp://en.wikipedia.org/wiki/Electrokinetic_phenomenahttp://en.wikipedia.org/wiki/Electric_fieldhttp://en.wikipedia.org/wiki/Electric_fieldhttp://en.wikipedia.org/wiki/Electric_fieldhttp://en.wikipedia.org/wiki/Interface_and_colloid_sciencehttp://en.wikipedia.org/wiki/Interface_and_colloid_sciencehttp://en.wikipedia.org/wiki/Interface_and_colloid_science


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Principles and procedure


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Horizontal gel electrophoresis

system


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Vertical gel electrophoresis

system


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Applications

1 DNA Analysis

2 Protein Analysis

3 Antibiotics Analysis

4 Vaccine Analysis


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Documents

Molecule Techniques