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Nong Lam University Department of Biotechnology

Thematic presentation:

MOLECULE TECHNIQUES

MSc. Ton Bao Linh

MOLECULE TECHNIQUES

Members
1. Hoang Thi Huyen Trang 11126237 DH11SH 2. Nguyen Thi Hong Phuc 3. Le Dang Huynh Tram 4. Le Nguyen Thao Ly 5. Le Nhat Tan 11126183 DH11SH 11126241 DH11SH 11126308 DH11SH 11126321 DH11SH

Sections
Hybridization PCR

Electrophoresis

Molecule techniques

Principle
The nucleic acid hybridization is the process where in two DNA or RNA single chains from different biological sources, make the double catenar configuration, based on nucleotide complementarity and of contingen sequence homology of the two sources, resulting DNA-DNA, RNA-RNA or DNARNA hybrids.

Principle
In most cases, the purpose of the hybridization techniques is identification or localization of certain nucleic acid sequences in the genome of some species. Two basic notions are used: the target molecule representing the DNA, RNA or protein sequence that should be identified and the probe molecule who identify the target, by hybridization.

Categories

Hybridization stages
1. Probe synthesis 2. Probe marking

3. Target DNA processing assumes the source

4. Target DNA denaturation

5. Target DNA transfer to

6. Molecular hybridization

Southern Blot

Edwin Mellor Southern

The original methodology

Stages

Modifications
First nylon membranes are less fragile than nitrocellulose sheets

Certain conditions the transferred DNA becomes covalently bound to the membrane during the transfer process

Nylon membranes efficiently bind DNA fragments down to 50 bp in length, where as nitrocellulose membranes are effective only with molecules longer than 500 bp

Applications and Limitations


The applications of Southern blotting are diverse and are not easily summarized in a short article. Two examples will suce to illustrate the range of research questions to which the technique can be applied.

Southern blotting in clone identification.

Restriction fragment length polymorphism analysis.

RFLP
In molecular biology, restriction fragment length polymorphism is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come 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 is broken into pieces (digested) by restriction enzymes and the resulting restriction fragments are separated according to their lengths by gel electrophoresis.

Applications
Analysis of RFLP variation in genomes was a vital tool in genome mapping and genetic disease analysis

RFLP analysis was also the basis for early methods of Genetic fingerprinting

Microarray
A microarray is a multiplex lab-on-a-chip. It is 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 antibody microarrays (also referred to as antibody matrix) in 1983 in a scientific publication and a series of patents

Applications
Help researchers to learn more about many different diseases including heart disease, mental illness and infectious diseases
Additionally, by examining the differences in gene activity between untreated and treated tumor cells.

The polymerase chain reaction (PCR) is a biochemical technology in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

History Developed in 1983 by Kary Mullis

In 1993, Mullis was awarded the Nobel


Prize in Chemistry along with Michael Smith for his work on PCR.

The method relies on thermal cycling, consist of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA

Components
DNA template that contains the DNA region (target) to be amplified. Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target. Taq polymerase or another DNA polymerase with a temperature optimum at around 70 C.

Components
Deoxynucleoside triphosphates (dNTPs, sometimes called "deoxynucleotide triphosphates)

Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
Divalent cations, magnesium or manganese ions (Mg2+, Mn2+)

PCR principles and proceduce

(1) Denaturing at 9496 C. (2) Annealing at ~65 C (3) Elongation at 72 C

PCR principles and proceduce

Figure 3: Ethidium bromide-stained PCR products after gel electrophoresis

PCR stages

Application of PCR

Detecting infectious agents

The role of PCR in cancer diagnostics

Genetic diseases and paternity testing

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.

Application of PCR

Amplification and quantification of DNA:


Real-time PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.

Electrophoresis
Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Ferdinand Frederic Reuss, who noticed that the application 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 of analytical techniques used in biochemistry for separating molecules by size, charge, or binding affinity.

Principles and procedure

Horizontal gel electrophoresis system

Vertical gel electrophoresis system

Applications
1 DNA Analysis

Protein Analysis

Antibiotics Analysis

Vaccine Analysis

References
http://www.hc-sc.gc.ca/sr-sr/biotech/aboutapropos/molecul_tech-eng.php

http://en.wikipedia.org/wiki/Polymerase_chai n_reaction
http://en.wikipedia.org/wiki/Electrophoresis

http://en.wikipedia.org/wiki/Hybridisation