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  • Digital Droplet PCR

    Diunggah oleh

    nuswantara
    33 tayangan21 halaman
    Droplet PCR is a third generation PCR following the invention of conventional PCR and Real-time PCR. The principle of this system relies on droplet generation into at least 20,000 nanodroplets. So, in addition to ordinary PCR, droplet PCR has an additional droplet generator in order to make this possible. This additional tool disperses an ordinary single PCR mix into literally 20,000 nano-PCR such that a rare event mutation will be visible with higher ratio than the non-droplet PCR

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    Droplet PCR is a third generation PCR following the invention of conventional PCR and Real-time PCR. The principle of this system relies on droplet generation into at least 20,000 nanodroplets. So, in addition to ordinary PCR, droplet PCR has an additional droplet generator in order to make this possible. This additional tool disperses an ordinary single PCR mix into literally 20,000 nano-PCR such that a rare event mutation will be visible with higher ratio than the non-droplet PCR

    Hak Cipta:

    © All Rights Reserved
    Unduh sebagai PDF, TXT atau baca online dari Scribd
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    33 tayangan21 halaman

    Digital Droplet PCR

    Diunggah oleh

    nuswantara
    Droplet PCR is a third generation PCR following the invention of conventional PCR and Real-time PCR. The principle of this system relies on droplet generation into at least 20,000 nanodroplets. So, in addition to ordinary PCR, droplet PCR has an additional droplet generator in order to make this possible. This additional tool disperses an ordinary single PCR mix into literally 20,000 nano-PCR such that a rare event mutation will be visible with higher ratio than the non-droplet PCR

    Hak Cipta:

    © All Rights Reserved
    Unduh sebagai PDF, TXT atau baca online dari Scribd
    Tandai sebagai konten tidak pantas
    dari 21

    Sukma Nuswantara

    Sukma Nuswantara@2017 1
    JENIS-JENIS PCR
    GENERASI 1

    CONVENTIONAL END POINT

    GENERASI 2

    PCR REAL TIME PCR FLUORESCENT

    GENERASI 3
    COMBINATION :
    DD-PCR END POINT &
    FLUORESCENT

    Sukma Nuswantara@2017 2
    DROPLET DIGITAL PCR (DD-PCR)
    - Amplifikasi dengan PCR biasa
    - Hasil PCR dibuat droplet berukuran nano dalam bentuk emulsi air-minyak
    sejumlah ± 20.000 nanodroplets = 20.000 PCR dalam satu tabung
    - Setiap droplet dilalukan dalam microchannel dan dianalisis dengan sinar
    laser

    Pembuatan
    nanodroplet

    PCR konvensional 20.000 nanodroplet


    (1 tabung) = 20.000 PCR
    (1 tabung)

    Sukma Nuswantara@2017 3
    PERANGKAT KERAS DIGITAL PCR
    1. PCR biasa
    2. Droplet maker
    3. Processor/reader
    4. Monitor display

    1 2 3 4

    Sukma Nuswantara@2017 4
    Sukma Nuswantara@2017 5
    Sukma Nuswantara@2017 6
    Sukma Nuswantara@2017 7
    • Kuantifikasi absolut: Tidak tergantung standar kalibrasi
    kuantitatif dan referensi (mis. Ct)
    • Lebih presisi : pengulangan PCR dalam 20.000 droplets/20 uL
    (target langka terdeteksi)
    • Signal-to-noise ratio: lebih tinggi, error lebih rendah
    • Toleransi lebih tinggi terhadap inhibitor
    • Mampu menganalisa campuran yang kompleks dan mendeteksi
    kontaminasi
    • Mendeteksi perubahan kecil secara linier

    Sukma Nuswantara@2017 8
    Sukma Nuswantara@2017 9
    Positive

    Threshold

    Negative

    Sukma Nuswantara@2017 10
    KONTAMINASI PADA
    SAMPEL : QC

    Contoh kontaminasi pada NTC (kiri) dengan amplitude


    sama dengan templat positif (kanan)

    Sukma Nuswantara@2017 11
    KUANTIFIKASI ABSOLUT

    Sukma Nuswantara@2017 12
    APPLICATION OF DD PCR

    •Liquid biopsy
    •Copy number variation (CNV)
    •Rare sequence detection
    •Gene expression and miRNA analysis
    •Single-cell analysis
    •Pathogen detection
    •Next-generation sequencing (NGS) library
    analysis

    Sukma Nuswantara@2017 13
    DETEKSI TARGET LANGKA
    (RARE EVENT DETECTION)

    1. Rare species detection:

    Contoh: deteksi viral genom dalam DNA manusia

    2. Rare Mutation Detection :

    Contoh: single-nucleotide mutation & deletion seperti dalam kasus-

    kasus penyakit kanker

    Sukma Nuswantara@2017 14
    PRINSIP RARE-EVENT DETECTION
    Reaksi PCR = 20uL

    Sampel dipartisi = 20.000 x 1 nanoliter

    40 droplet berisi mutan 19.960 droplet normal


    Kerapatan mutan = 33% (tanpa mutan)

    40.000 molekul normal


    40 mutan
    Kerapatan mutan = 0.1%

    Sukma Nuswantara@2017 15
    Catatan: Optimasi temperature (gradient) untuk panjang produk yang berbeda
    (mis. pada multipleks PCR) lebih mudah terlihat dibandingkan PCR biasa
    Sukma Nuswantara@2017 16
    Sukma Nuswantara@2017 17
    Sukma Nuswantara@2017 18
    Sukma Nuswantara@2017 19
    1. Studi biomarka dan CNV (Copy Number Variations)
    2. Pembuatan standar absolut molekuler
    3. Deteksi sekuen dan mutasi langka
    4. Deteksi Patogen
    5. Next Generation Sequencing
    6. Analisis ekspresi gen
    7. Analisis lingkungan
    8. Food testing
    9. dll

    Sukma Nuswantara@2017 20
    nuswantara@gmail.com

    Sukma Nuswantara@2017 21

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