Etiket Arşivleri: PCR

Polimeraz Zincir Reaksiyonu

POLİMERAZ ZİNCİR REAKSİYONU

• Polimeraz Zincir Reaksiyonu, DNA’nın belirli bir bölgesinin tüp içinde çoğaltılması işlemidir.

• Bu teknik ile DNA molekülünün milyonlarca hatta milyarlarca kopyasını yapmak mümkündür.

• PCR, 1985 yılında Cetus adlı biyoteknoloji firmasında çalışan Kary Mullis adlı araştırmacı tarafından bulunmuştur. Bu buluşu, Kary Mullis’in 1993 yılında Nobel ödülünü almasını sağlamıştır.

PCR,

• DNA dizi analizinde ve DNA haritalanmasında,

• Genetik hastalıkların teşhisinde,

• DNA parmakizi analizinde,

• İnsan Genom Projesi ve diğer Genom Projelerindeki araştırmalarda,

• Adli tıp örneklerinin genetik tiplendirilmesinde,

• Tarımda,

• Sistematik ve evrim çalışmalarında kullanılmaktadır.

• PCR tekrarlanan 3 basamak şeklinde gerçekleşir. Bu basamaklar;

1. Amplifiye edilecek DNA’nın yüksek sıcaklıkta denatürasyonu (94oC),

2. Primerlerin DNA üzerindeki hedef bölgelerle özgül hibridizasyonuna olanak verecek sıcaklıktaki annealing-birleşme reaksiyonu,

3. Taq DNA polimeraz enziminin yüksek aktivite gösterdiği sıcaklık olan 72oC’de primerlerin uzaması-tamamlayıcı zincir sentezi olarak tanımlanabilir.

Polimeraz zincir reaksiyonu için,

• Kalıp DNA molekülüne,

• 20-25 baz uzunluğunda ve kalıp zincirin 5’ve 3’ uçlarındaki DNA dizilerine komplementer olan sentetik oligonükleotitlere,

• Serbest dNTP’lere (dATP, dCTP, dGTP, dTTP)

• Taq DNA polimeraz enzimine

• Enzimin çalışacağı uygun koşulları sağlayan tampon sistemine ihtiyaç vardır.

Kaynak: http://yunus.hacettepe.edu.tr/~mergen/sunu/s_pcr.pdf

Current Status and Options for Biotechnologies in Food Processing and in Food Safety in Developing Countries ( FAO )

Acknowledgments
This background document is the result of the joint effort of many contributors. The initial key contributions were made by Olusola B. Oyewole from University of Agriculture, Abeokuta, Nigeria and Ruud Valyasevi from the National Center for Genetic Engineering and Biotechnology (Biotec),
Biotec, Bangkok, Thailand.
Sections relevant to biotechnology applications in food processing: Coordination was done by Rosa Rolle (Senior Agro-Industries and Post-harvest Officer, FAO Regional Office for Asia and the Pacific) who also made significant contributions to the document.
Sections relevant to biotechnology applications in food safety: Coordination was done by Masami Takeuchi (Food Safety Officer, Nutrition and Consumer Protection Division). Significantcontributions were made by Sridhar Dharmapuri, FAO consultant. Technical contributions from several FAO colleagues, including Maria de Lourdes Costarrica (Senior officer, Nutrition and Consumer Protection Division) and Annika Wennberg (Senior Officer, FAO JECFA Secretariat, Nutrition and Consumer Protection Division), to the document are also gratefully acknowledged. Comments from the following members of the ABDC-10 Steering Committee are gratefully acknowledged: Adama Diallo (Joint FAO/IAEA Division, Austria), Kathleen Jones (US Food and Drug Administration, United States), Marci Levine (International Life Sciences Institute, United States), Haruko Okusu (CGIAR Science Council, Italy), Masashi Kusukawa (Codex AlimentariusCommission, Italy) and Jørgen Schlundt (World Health Organization, Switzerland). Technical support and comments from John Ruane and Preet Lidder from the ABDC-10 Secretariat are also gratefullyacknowledged

E. Glossary of selected terms
Amplified Fragment Length Polymorphism (AFLP): Amplified fragment length polymorphism is a novel molecular fingerprinting technique that can be applied to DNAs of any source or complexity. Total genomic DNA is digested using two restriction enzymes. Double-stranded nucleotide adapters are ligated to the DNA fragments to serve as primer binding sites for PCR amplification. Primers complementary to the adapter and restriction site sequence, with additional nucleotides at the 3’-end, are used as selective agents to amplify a subset of ligated fragments. Polymorphisms are identified by the presence or absence of DNA fragments following analysis on polyacrylamide gels. This technique has been extensively used with plant DNA for the development of high-resolution genetic maps and for the positional cloning of genes of interest. However, its application is rapidly expanding in bacteria and higher eukaryotes for determining genetic relationships and for epidemiological typing.
Biosensor: is the self-contained analytical device that responds selectively and reversibly to the concentration or activity of chemical species in biological samples using various types of sensors of biological nature. Any sensor physically or chemically operated in biological samples can be considered as biosensor.
Classical mutagenesis: The process involves the production of mutants through the exposure of microbial strains to mutagenic chemicals or ultraviolet rays to induce changes in their genomes. Improved strains thus produced are selected on the basis of specific properties such as improved flavour-producing ability or resistance to bacterial viruses.
Conjugation: This is a natural process whereby genetic material is transferred among closely related microbial species as a result of physical contact between the donor and the recipient micro-organisms.
Enzyme-Linked Immunosorbent Assay (ELISA): An immunoassay that uses specific antibodies to detect antigens or antibodies in body fluids. The antibody-containing complexes are visualized through enzymes coupled to the antibody. Addition of substrate to the enzyme-antibody-antigen complex results in a coloured product.
Genomics: is the study of genes and their functions, and related technologies.
Hybridization: This is the common sexual breeding or mating process leading to genetic recombination. Such sexual crossing has led to offspring with superior or improved qualities. For example, the crossing of haploid yeast strains with excellent gassing properties and good drying properties could yield a novel strain with both good gassing and drying properties.
Polymerase Chain Reaction (PCR): is a method to amplify DNA in vitro, involving the use of oligonucleotide primers complementary to and annealing at different positions of nucleotide sequence in a target gene. The copying of target sequences is by the action of DNA polymerase.
Probe: is a single-stranded nucleic acid that has been radio-labelled, and is used to identify a complementary nucleic acid sequence that is membrane-bound.
Randomly Amplified Polymorphic DNA (RAPD): Randomly amplified polymorphic DNA and arbitrarily primed PCR (AP-PCR) represent novel DNA polymorphism assays, sometimes referred to as DNA fingerprinting, that involve the amplification of random DNA segments using PCR and oligonucleotide primers of arbitrary sequence. Products defining the polymorphisms exhibit Mendelian inheritance, and thus possess tremendous potential utility as genetic markers in a diverse array of scientific disciplines.
Recombinant DNA Technology: This technology is the application of in vitro nucleic acid techniques including recombinant deoxyribonucleic acid (DNA) and direct injection of nucleic acids into cells and organelles (adapted from Codex CAD/GL 44-2003, CAD/GL 45-2003, CAD/GL 45- 2003 and CAD/GL 68-2008)


Source: http://www.fao.org/docrep/meeting/019/k6993e.pdf

Polimeraz Zincir Reaksiyonu ( Koka ZONGO )

POLİMERAZ ZİNCİR REAKSIYONU(PZR)-POLİMERASE CHAIN REACTİON(PCR)

Koka ZONGO

I- PCR TARİHÇESİ VE TANINMI

II-DNA’NIN İÇERİĞİ VE YAPISI

III-PCR PRENSİBİ

IV-DÖNGÜNÜN AŞAMALARI

V. PCR REAKSİYON İÇİN GEREKLİ DUYAN ALETLERİ

VI.PCR ÇEŞİTLERİ

VII.PCR ANALIZI IÇIN UYGULAMA ÖRNEĞI

VII.PCR UYGULAMALARI

I- PCR TARİHÇESİ VE TANINMI

Polimeraz zincir reaksiyonu PZR İngilizce ’de Polymerase Chain Reaction yanı PCR adlı Amerika Bileşik Devletleri’nde 1984 ( 1984± 1) yılında Mullis et al. tarafından geliştirilen bir metottur.

Başlangıçta sadece DNA moleküler kopyasında bir metot olmuştur, dönem noktası hallettiren kişi Mullis Kary olmuş. Araştırmalarda büyük önem olduğu sebebiyle 1993 yılında Nobel kimya ödüllü oldu.

Hücre içerisinde küçük miktarıyla bulunan hedef DNA in vitro şekilde, bir enzim tarafından çoğaltılması bir tekniktir. Yani bu hücre’de hedeflenmış bir DNA parçası miktarı az olsa, PCR kullanılarak bu küçük DNA parçası çoğaltılabilirdir.

II-DNA İÇERİĞİ VE YAPISI

DNA genetik bilgiyi taşıdığı nükleotidlerin sekansında şifrelenmiştir. Nükleotidlerin yapısında bir baz, şeker ve fosfat bulunuyor. Bazlar ile ilgili adenin, guanin, sitozin ve timin mevcututur. Şeker ise β-D-2’-dezoksiribozdur.

Nükleotidler yan yana konarak bir zincir oluşur. Formül olarak dATP, dGTP, dCTP ve dTTP nüklotidler adı verir.

Bir DNA yapısında iki paralel zincir pürin (adenin ve guanin) baz ve pirimidin (sitozin ve timin) bazla hidrojen bağlantılardan bağlanmış ortaya çıktı.

Sunumunu bizlerle paylaşan üyemiz Koka ZONGO’ya teşekkürlerimizi sunarız.