Analisis DNA

Practice report

June 4, 2008

Fundamentals of  Fish Genetics

DNA Analyze

Gia Marta Novia

Abstract

The purpose this practice is knowing all about analyze DNA and identification zebra fish (D. rerio). Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. The main role of DNA molecules is the long-term storage of information. DNA is often compared to a set of blueprints or a recipe, since it contains the instructions needed to construct other components of cells, such as proteins and RNA molecules. The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information. The metodology of analyze DNA are three point and that is Extraction of DNA, PCR, and Electroforesis.  DNA extraction is a routine procedure to collect DNA for subsequent molecular or forensic analysis. The polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. Electroforesist is s method for knowing location of DNA. This method used to separation nukleic acid molecule as DNA component. The result this practice is : Absorbans = 0,814; Ratio = 1,723; Concentration DNA = 648 μg/ml; Protein = 0,0 mg/ml; Purity = 86 %. 

Interface

DNA is a long polymer made from repeating units called nucleotides. The DNA chain is 22 to 26 Ångströms wide (2.2 to 2.6 nanometres), and one nucleotide unit is 3.3 Å (0.33 nm) long. Although each individual repeating unit is very small, DNA polymers can be enormous molecules containing millions of nucleotides. For instance, the largest human chromosome, chromosome number 1, is approximately 220 million base pairs long.

Chemically, DNA is a long polymer of simple units called nucleotides, with a backbone made of sugars and phosphate groups joined by ester bonds. Attached to each sugar is one of four types of molecules called bases. It is the sequence of these four bases along the backbone that encodes information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA, in a process called transcription.

The purpose this practice is knowing all about analyze DNA and identification zebra fish (D. rerio).

Methodology

 This practice doing at Wednesday, April 7 till 31,  2008 in place seed development and fish genetic of laboratorium, Department of Aquaculture, Faculty of fissheris, Institut Pertanian Bogor.

Tools for used are micropipet, incubator, PCR, vortex, and oven. Whereas for component part such us cell lysis solution , proteinase K, RNAse, protein precipitation sollution, isopropanol, cold etanol 70%, SDW (Steryl Destilated Water), primer F actine, primer ß actine, dNTPs, Buffer Ex Taq, and Ex Taq, TBE, Etimidium Bromida and than TAE, loading buffer, and agarose 0,7 %.

The metodology of analyze DNA are three point and that is Extraction of DNA, PCR, and Electroforesis. At extraction of DNA choised fin fish zebra (Danio rerio) and than gived cell lysis solution 200 µL+1,5 proteinase K, incubation during overnight (55˚C). The next step, RNAse 1,5 ml and incubation again (1 hour, 37˚C). For getting sediment, used protein precipitation sollution 100µL and sentrifuse 12.000 rpm (15’) and for getting DNA sediment so need isopropanol 300 µL, sentrifuse 12.000 rpm in 10’. The next is gived cold etanol 70% 300 µL, sentrifuse again in 12.000 rpm 10’. After wet, mixed SDW (Steryl Destilated Water) 20 µL. The extraction of  DNA consist of PCR, denaturation, annealing, and extension.

At the next level metodology is PCR. Sample to PCR consist of premix. For make premix, have to mix SDW, primer F actine, primer ß actine, dNTPs, Buffer Ex Taq, and Ex Taq. Bactery enzyme which used for Ex Taq is from Thermos aquaticus. Electroforesis section began with TBE and Etimidium Bromida and than TAE, loading buffer, and agarose 0,7 %.

 Result and Describe

Picture 1. Result electroforesis (DNA in PCR) with UV ray

Absorbans = 0,814

Ratio = 1,723

Konsentrasi DNA = 648 μg/ml

Protein = 0,0 mg/ml

Purity = 86 %

Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. The main role of DNA molecules is the long-term storage of information. DNA is often compared to a set of blueprints or a recipe, since it contains the instructions needed to construct other components of cells, such as proteins and RNA molecules. The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information (Anonimous, 2008).

DNA extraction is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic steps in a DNA extraction, breaking the cells open to expose the DNA within, such as by grinding or sonicating the sample. Removing membrane lipids by adding a detergent. precipitating the DNA with an alcohol, usually ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet on centrifugation. This step also removes alcohol-soluble salt (Anonimous, 2008).

The polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be extensively modified to perform a wide array of genetic manipulations (Anonimous, 2008).

A basic PCR set up requires several components and reagents. These components include: DNA template that contains the DNA region (target) to be amplified. One or more primers, which are complementary to the DNA regions at the 5′ (five prime) and 3′ (three prime) ends of the DNA region. A DNA polymerase such as Taq polymerase or another DNA polymerase with a temperature optimum at around 70°C. Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand. Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis. Monovalent cation potassium ions(Anonimous, 2008).

Schematic drawing of the PCR cycle. Denaturing at 94-96°C,  Annealing at ~65°C, and  Elongation at 72°C. Four cycles are shown here. The blue lines represent the DNA template to which primers (red arrows) anneal that are extended by the DNA polymerase (light green circles), to give shorter DNA products (green lines), which themselves are used as templates as PCR progresses (Anonimous, 2008).

The purpose of a PCR (Polymerase Chain Reaction) is to make a huge number of copies of a gene. This is necessary to have enough starting template for sequencing. The fish which used for research is Danio rerio with clasification kingdom animalia, phylum Chordata, Class Actinopterygii, Order Cypriniformes, Family Cyprinidae, Genus Danio, Species D. rerio (Anonimous, 2008).

Electroforesist is s method for knowing location of DNA. This method used to separation nukleic acid molecule as DNA component. Nukleic acid to separated by electric current, usually on solid media. The electric current gived between two electrode causes DNA move from negative pole to positive pole (Permana, 2008).

The result this practice is : Absorbans = 0,814; Ratio = 1,723; Concentration DNA = 648 μg/ml; Protein = 0,0 mg/ml; Purity = 86 %.

Conclusion 

There are three point metodology of analyze DNA. That is extraction of DNA, PCR, and electroforesis. Schematic drawing of the PCR cycle. Denaturing at 94-96°C,  Annealing at ~65°C, and  Elongation at 72°C. Four cycles are shown here. The blue lines represent the DNA template to which primers (red arrows) anneal that are extended by the DNA polymerase (light green circles), to give shorter DNA products (green lines), which themselves are used as templates as PCR progresses. The result this practice is : Absorbans = 0,814; Ratio = 1,723; Concentration DNA = 648 μg/ml; Protein= 0,0 mg/ml; Purity= 86%.

 

 

Refference

 Anonimous. 2008. DNA. www.wikipwedia.org. [May, 12, 2008].

Anonimous. 2008. DNA extraction. www.wikipwedia.org. [May, 31, 2008].

Anonimous. 2008. Polymerase chain reaction. www.wikipwedia.org. [May, 12, 2008].

Anonimous. 2008. Zebrafish. www.wikipwedia.org. [May, 31, 2008].

Permana. 2008. PCR untuk diagnosa penyakit ikan. www.aodoyz.blogspot.com. [April, 12, 2008].

Pratikum kali ini bertujuan untuk mengetahui tentang bagaimana menganalisis DNA dan mengetahui jenis ikan dari jaringan ikan.

About Gya's Blog

"Gya" Mahasiswi IPB (2007)...Mayor Teknologi dan Management Perikanan Budidaya...

Posted on 08/01/2012, in Sains. Bookmark the permalink. 2 Komentar.

  1. nice blog,,so colorfull..
    ternyata adek kelas🙂

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