SMARTer 시리즈

SMARTer 시리즈 SMARTer Solutions 2014.12.17 다카라코리아바이오메디칼

SMART 하게실험하고 SMARTer 한결과를얻는방법 Clontech SMARTer 시리즈

Contents 1. Introduction 1. 역전사반응이란? 2. 기존역전사반응의한계 2. SMARTer 시리즈 1. SMARTer 란? 2. SMARTer 시리즈와적용 3. 현재진행형 (~ing) 이벤트

Central Dogma ( 분자생물학의중심원리 ) RNA DNA General Special (Virus) by Francis Crick in 1958 General Special Unknown DNA DNA RNA DNA protein DNA DNA RNA RNA RNA protein RNA RNA protein DNA protein protein protein 그림출처 : Wikipedia

역전사반응 (cdna 합성 ) 조직 Total RNA mrna 추출 Reverse transcription RNAiso Plus/Blood Takara MiniBEST 시리즈 cdna ; Complemantary DNA Reverse Transcription (cdna synthesis) 1. Central dogma의역과정 (RNA DNA:Reverse Transcription) 2. 왜 cdna를합성하여연구하는가? - RNA는 DNA에비하여불안정하다. - Total DNA에는 Exon과 intron이모두포함 - 유전자량증폭을위하여 DNA 합성이필요 (PCR Polymerase=DNA dependent DNA polymerase)

Reverse Transcriptase ( 역전사효소 )? Reverse Transcriptase (RTase)? - RNA-dependent DNA polymerase - RNA 로부터 complementary DNA (cdna) 를합성하는효소 RTase 의기본특성 (a) RNA-dependent DNA polymerase (b) ribonuclease H (c) DNA-dependent DNA polymerase Crystallographic structure of HIV reverse transcriptase RTase 의종류 MMLV RTase AMV RTase 유래 Moloney murine leukemia virus (M-MLV) Avian myeloblastosis virus (AMV) - HIV-1 (human immunodeficiency virus type 1) reverse transcriptase - 용도 M-MLV (Moloney first strand murine cdna leukemia synthesis virus) reverse transcriptase first strand cdna synthesis - AMV (avian myeloblastosis virus) reverse transcriptase 반응온도 37 42 Research에서주로사용하는효소 - Telomerase reverse transcriptase RNaseH RNaseH 활성이낮다 RNaseH 활성이높다 기타 DNA endonuclease 과 RNaseH 활성이낮아 AMV 에비해증폭효율이높고긴 cdna 를합성 고차구조 RNA 증폭에적합하며, 활성도가높아 MMLV 에비해고가 출처 : Wikipedia

기존 cdna 합성의한계 (start) (stop) 5 AAAAA 3 mrna transcript (poly A + RNA) RNA 전처리 DNase 처리, mrna 정제 mrna 비율 : 0.5~2% Second Strand Enzyme Cocktail RNase H DNA pol DNA Ligase 5 AAAAA 3 Prime mrna with oligo primer RT extends primer 3 TTTTT 5 5 3 oligo primer AAAAA RNase H creates nicks in RNA TTTTT 5 말단 cdna 합성이어려움 Full length cdna 합성률낮음 복잡한 2nd strand cdna 합성 AAAAA TTTTT AAAAA TTTTT DNA pol extends 3 ends, displacing next RNA piece Ligase ligates segments Adaptor 부착을위한 Ligation 과정 Gubler and Hoffman procedure (Gubler, U. & Hoffman, B. J. (1983) Gene 25:263-269)

이럴때, SMARTer 하세요 RNA를추출, 연구하는고객 cdna 합성고객 Library를제작하는고객 Next Generation Sequencing (NGS) 고객 고객께질문하세요. cdna 합성만족하세요? Checking List! 고객이아래와같이고민한다면.SMARTer Full length cdna를얻기가너무힘들어요. 5 말단까지합성이안되네요. 2nd strand cdna 합성을또? 너무번거롭고귀찮아요. cdna 합성전, 처리과정이너무번거롭고많아요! (DNase 처리, mrna 정제등 ) Adaptor ligation을해야하는데 Ligation 효율이너무좋지않아요. 이런문제, 한번에해결할수없나요?

SMARTer 가왜좋은가? Clontech SMARTer 는,, Simple Powerful Quality * 1~2ng의소량의 total RNA로고품질의 cdna 합성 * 풍부한 Full length cdna 획득 * Ligation과정없이고효율로 Adaptor 부착 * Single Step: RNA sample의 loss, 오염등방지 & 간편

SMARTer 기술? Full length cdna 를합성하기위한 Clontech 의독자적인기술 - SMART: Switching Mechanism At 5 -end of RNA Transcript - 한발더나아간 SMART Better, SMARTer SMARTer 역전사효소의특징 1. Terminal Transferase activity ( 말단전이효소 ) ability to add nucleotides to the 3 terminus of cdna 3 nnnnnngcccagcatggttac..5 2. Template switching activity ( 주형전환활성 ) changing templates, jumping to different mrna templates - 변형 MMLV 역전사효소 (Moloney Murine Leukemia Virus RTase) -SMARTer 역전사효소의특징 1. Terminal Transferase 2. Template Switching..CCGUUACG GCAAUCGUUAAGGU..

SMARTer 기술의원리 1 Starting material Modified Oligo dt primer Terminal Transferase 활성 2 Single strand cdna Template Switching 활성 PCR 증폭 Adaptor 서열을이용한 PCR 증폭 3 Double strand cdna (Full length)

SMARTer 시리즈 <SMARTer 시리즈 > 목적에따라다양한 SMARTer cdna 합성키트 제품명 Code* 1 SMARTer PCR cdna Synthesis Kit 634925 SMARTer Pico PCR cdna Synthesis Kit 634928 2 SMARTer RACE 5'/3' Kit 634858 1 일반 cdna 합성 5 Next Generation Sequencing 3 In-Fusion SMARTer Directional cdna Library Construction Kit 634933 SMART cdna Library Construction Kit 634901 2 RACE 3 Library 구축 4 Substraction 4 Clontech PCR-Select cdna Substraction Kit 637401 5 SMARTer Ultra Low Input RNA Kit for 634848 Sequencing - v3 외 * 가장작은용량의코드만나타냄

1. SMARTer 시리즈와적용 (cdna 합성 ) Q1. Full length 의 cdna 를합성해서 Cloning, PCR 등에사용하려면? SMARTer PCR cdna Synthesis Kit (Code 634925) SMARTer Pico PCR cdna Synthesis Kit (Code 634928)

SMARTer 와 In-Fusion 이만나면?

SMARTer 시리즈 <SMARTer 시리즈 > 목적에따라다양한 SMARTer cdna 합성키트 제품명 Code* 1 SMARTer PCR cdna Synthesis Kit 634925 SMARTer Pico PCR cdna Synthesis Kit 634928 2 SMARTer RACE 5'/3' Kit 634858 1 일반 cdna 합성 5 Next Generation Sequencing 3 In-Fusion SMARTer Directional cdna Library Construction Kit 634933 SMART cdna Library Construction Kit 634901 2 RACE 3 Library 구축 4 Substraction 4 Clontech PCR-Select cdna Substraction Kit 637401 5 SMARTer Ultra Low Input RNA Kit for 634848 Sequencing - v3 외 * 가장작은용량의코드만나타냄

2. SMARTer 시리즈와적용 (RACE) Q2. RACE 분석을하고싶어요. 미지의서열을분석목적으로 cdna 합성하려면? SMARTer RACE 5'/3' Kit (Code 634858) Rapid Amplification of cdna Ends (RACE) 알고있는유전자서열을중심으로 5 방향, 3 방향의미지의유전자서열의 cdna 를합성하여분석하는방법 RACE cdna 합성후, PCR 분석, Sequencing 등여러실험에적용 5 end Known gene 3 end 5 RACE 3 RACE

SMARTer RACE 5 /3 Kit Product Feature Input type & amount range : 10 ng 1μg or total or poly A+RNA Input volume : Up to 10 μl input volume Complete kit: Includes a pre-linearized vector & In-Fusion HD system to clone RACE fragments Benefit 다양한초기샘플량적용가능 초기샘플 volume 증가 : 희석된샘플도적용가능 ( old version : 3.5 μl) Better value : old version 은 DNA polymerase (Advantage 2) & cloning system 별도준비 Component SMARTer RACE 5 /3 Kit Components SeqAmp DNA Polymerase In-Fusion HD Cloning Kit New linearized prace vector NucleoSpinGel and PCR Clean-Up kit Stellar Competent Cells RACE cdna 합성부터, Library 제작을위한 In-Fusion Cloning 까지! 17

Tip. 함께추천하면좋은제품 RACE OK! Library OK! In-Fusion reaction Linearized vector Recombinant vector Day 1 Day 2

SMARTer 시리즈 <SMARTer 시리즈 > 목적에따라다양한 SMARTer cdna 합성키트 제품명 Code* 1 SMARTer PCR cdna Synthesis Kit 634925 SMARTer Pico PCR cdna Synthesis Kit 634928 2 SMARTer RACE 5'/3' Kit 634858 1 일반 cdna 합성 5 Next Generation Sequencing 3 In-Fusion SMARTer Directional cdna Library Construction Kit 634933 SMART cdna Library Construction Kit 634901 2 RACE 3 Library 구축 4 Substraction 4 Clontech PCR-Select cdna Substraction Kit 637401 5 SMARTer Ultra Low Input RNA Kit for 634848 Sequencing - v3 외 * 가장작은용량의코드만나타냄

3. SMARTer 시리즈와적용 (NGS) Clontech SMARTer 는,, * 피코그람수준의 RNA 로부터 cdna 합성 * Full length 의 cdna 획득 * Ligation 과정없이 Adaptor 부착 ( 고효율 ) Simple Powerful Quality 극소량 (Low Input) Sample 로부터 NGS 용 Library 제작에최적! 다수의 NGS Platform 이인정한최고의제품 일반 SMARTer cdna 합성제품도 NGS 고객에게추천가능! SMARTer PCR cdna Synthesis Kit (code 634925, 634926) SMARTer RACE 5'/3' Kit (code 634858, 634859)

3. SMARTer 시리즈와적용 (NGS) Q3. Next Generation Sequencing 용으로 cdna 를합성하고싶어요. SMARTer Ultra Low Input RNA Kit for Sequencing v3 최고의민감도 : 1-1,000 cells, 10 pg 10 ng mrna 시간단축 : Purification 과정감소 (Single SPRI-bead purification of ds cdna) cdna 증폭효율향상 : SeqAmp DNA Polymerase NGS 기기적용성 : Illumina and Ion Torrent High-quality RNA-seq data

SMARTer for NGS 선택가이드 ( 샘플별 ) NGS workflow 샘플시료 Starting sample rrna 제거 SMARTer cdna 합성 Library 제작 Library 정량 NGS 분석 mrna (PolyA+) SMARTer Ultra Low Input-v3 RNA Single Cell Total RNA RiboGone SMARTer Ultra Low Input-FluidigmC1 SMARTer Universal Low Input Library Prep Library Quantific ation kit (for illumina) NGS 분석 -Illumina, -Ion torrent SMARTer stranded RNA-Seq DNA ChIP DNA DNA SMART -ChIP Seq

SMARTer for NGS 선택가이드 ( 기기별 ) 아래의기기를보셨다면! 제품추천은이렇게! Illumina MiSeq, HiSeq. Ion torrent PGM Fluidigm C1 PacBio RSII Single cell & mrna Ultra Low Input-v3 Ultra Low Input-v3 Ultra Low RNA Kit for the Fluidigm C1 Total RNA Universal Low Input Stranded RNA-Seq Universal Low Input cdna Synthesis kit (634925, 634926) ChIP DNA DNA ChIP-Seq Kit Library quantification Kit

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