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Fluoresans Ve Moleküler Biyolojide Uygulamalari

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Fluoresans Ve Moleküler Biyolojide Uygulamalari FLUORESANS VE MOLEKÜLER BİYOLOJİDE UYGULAMALARI Prof.Dr. Nermin Gözükırmızı İstanbul Üniversitesi Fen Fakültesi, Moleküler Biyoloji ve Genetik Bölümü FLUORESANS Bir atom veya molekülden elektromagnetik enerjinin absorbsiyonunu izleyen bir süre sonucu dışarıya yayılan ışımadır. Görünebilir ışık spektrumu: Fluoresans sınırları Kalsiyum Fluorit CaF2 Fluoresans(Fluoresans, molekülün belli dalga boyundaki ışığı absorblaması ve bunu tekrar daha büyük bir dalga boyunda yayması olayıdır). Luminesans (Bir dış kaynaktan alınan enerjinin bir kısmının elektro manyetik ışınım olarak salmasıdır). Bazı tanımlar • Emilim – Işık bir objenin içinden geçtiğinde değişen oranlarda ışığı absorblar. Böylece absorblanan ışığın dışındaki dalga boylarında renk üretilir. • Kırılma – Işığın bir optik yoğunluğa sahip ortamdan farklı optik yoğunluktaki bir otama geçişindeki yön değişimidir. • Yansıma – Işık ışınları bir cismin etrafında yön değiştirmesidir. Yansımasıdır. • Dağılma – Işık şeffaf bir alana girdiğinde, ışığın onu oluşturan farklı dalga boylarına ayrılması. Kontrol Absorpsiyon Yeşil ve mavi yok Kırmızı filtre Işık absorbsiyonu Beyaz ışık Mavi ışık Kırmızı ışık Yeşil ışık Kırmızı ve yeşil Mavi ve yeşil Mavi ve kırmızı absorblanır absorblanır absorblanır Absorbsiyon şeması Beyaz ışıktaki Absorblanan ışığın rengi renk kırmızı mavi yeşil mavi kırmızı yeşil yeşil kırmızıkırmızı mavi sarı mavi mor yeşil cyan kırmızı siyah kırmızı yeşil mavi gri pembe yeşil mavi • Kromoforlar ışığı absorblayan molekül bileşenleridir. • Örneği proteinlerde indol halkası benzeri. • Genellikle aromatik halkalardır. Jablonski Diagramı S’ 1 S1 hvex hvem S 0 Fluorescein – Tipik Fluoresan Prob 120 100 80 60 Intensity 40 20 0 380 480 nm 580 680 NEDEN FLUORESANS? • Çok güzel. • Moleküler çevre hakkında bilgi verir. • Dinamik olaylarda nanosaniye düzeyinde bilgi verir. The Nobel Prize in Chemistry 2008 "for the discovery and development of the green fluorescent protein, GFP" Osamu Shimomura (1962) Martin Chalfie Roger Y. Tsien 1/3 of the prize 1/3 of the prize 1/3 of the prize USA USA USA Marine Biological Laboratory (MBL) Columbia University University of California Woods Hole, MA, USA New York, NY, USA San Diego, CA, USA b. 1928 b. 1947 b. 1952 pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP) GFP • 1962 bulunması • 1992 klonlanması Prasher • 1994 ilk gen anlatımı uygulaması Chalfie et al. • 1996 kristal yapısının bulunması Düşük toksisiteli, yüksek stabiliteli monomerik kromofor. GFP kullanım alanları • Gen anlatımının görülmesi • Protein etiketleme, hareketi, ilişkileri,aktivite ölçümü ve yıkımı • Organel etiketleme, füsyon vb. • Genetik sensörler pH, Ca+2, cAMP, cGMP,kinaz aktivitesi, voltaj vb. Osamu Shimomura Douglas Prasher Marty Chalfie Sergey A. Lukyanov Roger Tsien Shimomura (1979) In June 2003 GFP was the protein databank's (pdb) molecule of the month Işıma deniz suyundaki Ca+2 iyonları varlığında gerçekleşir. GFP, kendi kromoforunun oluşumunu katalizler. Bu katalizde Arg96’nın çok önemli bir rol oynadığı önerilmiştir. Douglas Prasher GFP Amino Acid Sequence: MSKGEELFTGVVPVLVELDGDVNGQKFSVSGEGEGDATYGKLTLNFICT TGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTI FYKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKMEYNYNS HNVYIMGDKPKNGIKVNFKIRHNIKDGSVQLADHYQQNTPIGDGPVLLP DNHYLSTQSALSKDPNEKRDHMILLEFVTAARITHGMDELYK GFP E.coli, Chalfie. Fluoresan timer protein Dsred (2000) Sergey A. Lukyanov 44 yeni veya mutant variant GFP kristal yapısı GFP uzun bir zincir boyunca birleşen 238 aminoasit içerir. Bu zincir bir fıçı biçiminde katlanır. Fıçı yapısının içinde 65., 66. ve 67. aminoasitler UV ve mavi ışığı absorplayan kimyasal grupları oluşturur, ve yeşil fluoresan ışık yayar. GFP ışığı absorblayan ve yayan bir kimyasal grup olan kromofor taşır. Fluoresan proteinler Aequorea fluoresan protein (AFP) varyantları Yeşil fluoresan protein (GFP) Arttırılmış yeşil fluoresan protein (EGFP)508nm Sarı fluoresan protein (YFP) Arttırılmış sarı fluoresan protein (EYFP) 528nm Cyan fluoresan protein (CFP) Arttırılmış cyan fluoresan protein (ECFP)475nm Mavi fluoresan protein (BFP) Arttırılmış mavi fluoresan protein (ECFP)448nm Kırmızı fluoresan proteinlerin bulunması Anthozoa Yeni yeşil fluoresan benzeri proteinler Copepoda Fluorescence shown along the body structure of amphioxus. Credit: Scripps Institution of Oceanography at UC San Diego May 26, 2009—Fluorescent proteins in some sea creatures may actually promote a healthy glow, according to a new study of a fishlike animal called a lancelet (pictured). in vivo görüntüleme tumor (GFP)vasculature (Angiosense) Brainbow mice Painted in Fluorescent Serebral korteks Bacteria • Sarı hücreler replikasyonun başladığını, yeşil hücreler sentez (S) fazındaki ve kırmızı hücreler ise G1 fazındaki hücreleri işaret etmekte. • Fucci (fluorescent, ubiquitination-based cell cycle indicator). 46 Oksidatif Reaksiyonlar • Superoksid Hidroethidin • Hidrogen Peroksid Diklorofluorescein • Glutathion seviyesi Monobromobiman • Nitrik Oksid Diklorofluorescein Nükleik asid probları • Hoechst 33342 (AT rich) (uv) 346 460 • DAPI (uv) 359 461 • POPO-1 434 456 • YOYO-1 491 509 • Acridine Orange (RNA) 460 650 • Acridine Orange (DNA) 502 536 • Thiazole Orange (vis) 509 525 • TOTO-1 514 533 • Ethidium Bromide 526 604 • PI (uv/vis) 536 620 • 7-Aminoactinomycin D (7AAD) 555 655 Protein probları Probe Excitation Emission FITC 488 525 PE 488 575 APC 630 650 PerCP™ 488 680 Cascade Blue 360 450 Coumerin-phalloidin 350 450 Texas Red™ 610 630 Tetramethylrhodamine-amines 550 575 CY3 (indotrimethinecyanines) 540 575 CY5 (indopentamethinecyanines) 640 670 Moleküler yapı ve Hücre organizasyonu dinamiği ve dinamiği Geliştirilmiş yüzeyler Hayvansal sistemler İşlevsel genomik uygulamaları – İnsersional mutagenez – Enhancer tanısı – Reporter gen anlatımı • Hücre tanısı • Canlı hücrelerde protein trafiği ve hücre yapıları – Füsyon proteinler • protein-protein ilişkileri • Hücre içi iletişim –Fotoaktif ve foto dönüşümlü fluoresan proteinler. • Hayvan transformasyon sistemleri –Promotörler • Biyo-ekoloji – Etikletli spermler • Canlı hücreler ve bölümlerinde biyokimyasal analizler – Biosensörler • Protein fotoinaktivasyonu ve in vivo hücre öldürülmesi The Nobel Prize in Chemistry 1980 for his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant-DNA" "for their contributions concerning the determination of base sequences in nucleic acids" Paul Berg Walter Gilbert Frederick Sanger 1/2 of the prize 1/4 of the prize 1/4 of the prize USA USA United Kingdom Harvard University, Biological MRC Laboratory of Stanford University Laboratories Molecular Biology Stanford, CA, USA Cambridge, MA, USA Cambridge, b. 1926 b. 1932 United Kingdom b. 1918 DNA analizleri elektroferogram 2008’in en iyi icadı evde yapılan DNA testi hürriyet.com.tr 8 Kasım 2008 Monday, November 10, 2008 1. The Retail DNA Test TIME's Best Inventions of 2008 Time Dergisi yılın en iyi 50 icadını seçti. Dergiye göre, 2008’in en iyi icadı, bu yıl perakende olarak satışa sunulan pratik DNA testi. 399 dolara satın alınabilen paket sayesinde herkes ev ortamında analiz yaparak, genetik olarak 90 hastalık ve rahatsızlığa yakalanma riskini öğrenebiliyor. Test paketi, 600 bin gen kombinasyonunu tanıyıp yorumlayarak, kişinin Alzheimer’a yakalanma ihtimalinden, kel kalma veya kör olma riskine dek birçok konuda tıbbi olasılık bilgisi veriyor. "23andMe" adlı testi pazarlayan firmanın sahibi, Google’ın kurucularından Sergey Brin’in eşi Anne Wojcicki. •Age-related Macular Degeneration •Alcohol Flush Reaction •Bitter Taste Perception •Celiac Disease •Crohn's Disease •Cystic Fibrosis (Delta F508 mutation) •Earwax Type •Eye Color •G6PD Deficiency •Lactose Intolerance •Malaria Resistance (Duffy Antigen) •Muscle Performance •Age-related Macular Degeneration •Alcohol Flush Reaction •Bitter Taste Perception •Celiac Disease •Crohn's Disease •Cystic Fibrosis (Delta F508 mutation) •Earwax Type •Eye Color •G6PD Deficiency •Lactose Intolerance •Malaria Resistance (Duffy Antigen) •Muscle Performance •Non-ABO Blood Groups •Norovirus Resistance •Parkinson's Disease •Prostate Cancer •Psoriasis •Resistance to HIV/AIDS •Rheumatoid Arthritis •Sickle Cell Anemia & Malaria Resistance •Type 1 Diabetes •Type 2 Diabetes •Venous Thromboembolism Yeni dizileme yöntemleri (Yeni genom center görünümü) Roche/454 GS FLX Dizileme hazırlığı Tüm genom rastgele parçalara ayrılır Parçalar nötralize edilir. Adaptörler eklenir. DNA su yağ karışımındaki tutucu boncuklara bağlanır. Boncuklara bağlı parçalarda PCR yapılır. Bir parçanın binlerce kopyasını içeren boncuklar PicoTiter Plate’te konur. Polimeraz ve luciferaz içeren enzimler eklenir. Dizileme işlemi Plate’ler dizileme aygıtına konur. Nukleotidler (A,C,G,T) plate üzerinde seri halinde yıkanır. Tamamlayıcı nukleotid girince, DNA polimeraz ile kalıp DNA uzatılır. Nukleotidlerin eklenmesi ışık saçar ve bu durum CCD kamera ile okunur. Binlerce boncuk ayni anda Genome sequencing in microfabricated high- dizilenir. density picolitre reactors-Nature 437, 376-380 (15 September 2005) Dizileme hızı • ~25 milyon baz >=% 99 doğrulukta 4 saatte • ~230,000 okuma • Ortalama okuma uzunluğu 110 baz Günümüzde kullanılan yeni nesil dizileme sistemleri Roche 454 Illumina HiSeq Helicos Heliscope Solid dizileme Ion Torrent Dizileme Pasific Bioscience Pacific Biosciences Tek Molekül Real Time (‘Single Molecular Real Time –SMRT) teknolojisidir. Her bir çip, 100 nm boşlukta DNA izlenir. Fosfor ile işaretli nükleotidler farklı renkle işaretlenir. Uzun okumalar, kısa sürelerde okunur ve yüksek
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