cDNA 말단의 신속 증폭
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Overview
출처: 파블로 산체스 보쉬2,숀 코코란2, 카타 브뤼크너1,2,3
1 엘리와 에디테 브로드 재생 의학 및 줄기 세포 연구 센터
2 세포 및 조직 생물학학과,
3 미국 캘리포니아 주 샌프란시스코 대학교 심장혈관 연구소
cDNA 끝(RACE)의 신속한 증폭은 시퀀스에 대한 사전 지식 없이도 3′ 또는 5’끝으로 확장하여 mRNA로부터 전신 cDNA를 증폭할 수 있는 기술이다(Frohman et al., 1988). 일반 PCR과는 달리, 그것은 단지 하나의 특정 PCR 프라이머와 대부분의 mRNA에 무차별 적으로 바인딩됩니다 두 번째 비 특이적 프라이머를 사용합니다. 증폭되는 영역이 mRNA의 3′ 또는 5’끝에 있는지 여부에 따라, 두 번째 프라이머는 모든 성적증명서(5’end)에 추가된 폴리A 꼬리(3’end) 또는 합성 링커를 결합하도록 선택된다. 이러한 프라이머 조합은 한쪽-3 또는 5′(오하라 외, 1989)의 공지된 시퀀스를 사용하여 PCR 증폭으로 인해 “일방적” 또는 “고정된” PCR을 산출하는 것으로 알려져 있다. 접근은 그렇지 않으면 검출하기 어려울 유전자 특정 희귀 mRNAs의 포착을 허용합니다, 예를 들면 그들의 상대적으로 낮은 발현 또는 알려지지 않은 완전한 순서 때문에 (Frohman et al., 1988).
RACE는 mRNA로부터 단일 좌초 된 무료 DNA (cDNA)를 합성하기 위해 역 전사 단계 (RT)로 시작됩니다. 이것은 관심있는 유전자에서 cDNA 단편을 증폭시키는 2개의 연속PCRs에 선행됩니다. RACE를 수행하기 위해서는, 관심 있는 유전자의 서열은 유전자 특이적 프라이머(GSPs)를 설계하는 데 필요한 바와 같이 적어도 부분적으로 알려져야 합니다. 프로만, 1994년; 리우와 고로프스키, 1993년). 두 번째 프라이머 쌍은 샘플에 존재하는 모든 성적체에 대한 음침을 줄이는 일반 프라이머이기 때문에 PCR 반응의 특이성이 감소됩니다. 따라서 RACE는 비특이적 성적증명서를 증폭할 확률을 낮추기 위해 중첩된 두 개의 PCR로 이상적으로 수행됩니다(그림 1). GSP에 비해 증폭방향에 따라 이 기술은 5′ 또는 3’RACE(도 1)로 분류됩니다.
3′ RACE(도 1A)는 mRNA에 존재하는 폴리(A) 꼬리를 비특이적 프라이머에 대한 제네릭 결합 부위로 활용한다. 이것은 oligo (dT) 프라이머를 사용하여 cDNA를 합성 할 수 있기 때문에 기술을 단순화합니다. GSP는 성적 증명서의 5’영역에 위치하고 있습니다. 이 RACE 변종은 성적 증명서 변형및 다른 3’UTR (스코토 라비노 등, 2007a)의 검출을 허용합니다. 5’RACE (도 1B)에서, GSPs는 유전자의 3’영역에 위치합니다. 모든 성적증명서에 결합하는 비특이적 프라이머를 사용할 수 있도록 일반 프라이머 결합 부위역할을 하는 어댑터가 5’RNA 끝에 부착된다. mRNA에 어댑터를 부착하려면 외핵체로부터 mRNA를 보호하고 번역을 촉진하는 5’캡을 제거해야 합니다(Bird et al., 2016). 3’RACE에 반대, 5’RACE는 차동 5의 스플라이스 변형과 대안 5’UTR (스코토 라비노 등, 2007b)를 찾는 데 도움이됩니다.
여기에서 는 드로소필라 dSmad2 (Smox)유전자에 의해 인코딩된 공지된 5′ 서열(그림 2A)을 사용하여 상이한 성적증명서 변이체를 식별하고 격리하기 위해 3’RACE를 수행합니다. dSmad2, 플라이 스마드 단백질, 액티빈 β 신호의 중요 한 변환기, TGF-β 슈퍼 패밀리의 경로. dSmad2는 세포 증식, 세포 세포 증식 및 분화와 같은 다중 세포 과정을 조절합니다(Upadhyay et al., 2017). dSmad2의 차분하게 접합된 성적증명서가 성인 비행에서 다른 기능을 가질 수 있기 때문에, 이러한 잠재적 기능을 탐구하는 첫 번째 단계는 이 발달 단계에서 dSmad2의 모든 성적 증명서 변형을 평가하는 것입니다.
Procedure
Results
There are two annotated transcripts for Drosophila dSmad2 in FlyBase (Fig. 2A). Our results, however, reveal three different transcripts for dSmad2, ranging in size from 750 bp to 1400 bp (Fig. 2B). Differences in the intensity of the bands indicate their relative expression levels. Among the predicted products, one transcript is predominant (Fig. 2B, black arrow A), and one is expressed at a lower level (Fig. 2B, black arrow B). In addition, a previously undescribed smaller product was detected (Fig. 2B, grey arrow C).
The identification of such rare transcripts is only possible with a sensitive method such as RACE. Following RACE, one can clone the fragments obtained by PCR to study the transcript sequence, find its similarity with other transcript variants and clone it for transgenesis. Such experiments help investigate the functions of transcript variants specific to a tissue or developmental stage.
Figure 1. Schematics of the two different RACE approaches. A) 3’ RACE. cDNA is synthesized by using oligo (dT) primers. The same oligo (dT) primer is used in combination with a 5’ gene-specific primer to amplify 3’ cDNA ends through one, or better two, rounds of PCR. B) 5’ RACE. RNA is first decapped to free the 5’ end. In a second step, an adapter RNA sequence is added to the 5’ end. cDNA is generated by using a primer complementary to the adapter sequence. The same primer is used in combination with 3’ gene-specific primer/s to amplify the cDNA.
Figure 2. A) RACE allows amplification of several transcripts from the same gene, exemplified by 3’ RACE of Drosophila dSmad2. Combination of a nonspecific primer (Oligo dT) with a gene-specific primer (GSP) yields cDNAs of different lengths (Product A, Product B) corresponding to alternatively spliced transcripts (mRNA A, mRNA B). B) Separation of PCR products of dSmad2 3’ RACE on a 1% agarose gel. Lanes correspond to (1) 1 kb DNA ladder as marker, (2) no RT negative control (contains RNA and primers), (3) no primers negative control (contains cDNA template) (4) full reaction of 3’ RACE for dSmad2 (contains. primers and cDNA template). Amplification of dSmad2 transcripts by 3’ RACE yields three distinct products (arrows). Two cDNAs were of predicted sizes around 1400 and 1200bp (black arrows A and B, corresponding to Product A and B in (A)). In addition, a previously undescribed smaller cDNA of ~750bp was detected (grey arrow, C).
Applications and Summary
RACE provides a quick, inexpensive and powerful tool to obtain cDNAs from sequences that are only partially known, or from rare transcripts that are otherwise harder to amplify. It can be used to either find the sequence of unknown transcripts from an already known gene or to clone such transcripts for further studies. Following RACE, such transcripts can be overexpressed in cell-based systems or model organisms to investigate their function in vivo.
Acknowledgements
This work was supported by grants from the National Science Foundation # IOS-1355222 and the National Institutes of Health # 1R01GM112083 and 1R01GM131094 (to K.B.).
References
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- Frohman, M.A. (1994). On beyond classic RACE (rapid amplification of cDNA ends). PCR Methods Appl. 4, S40–S58.
- Frohman, M.A., Dush, M.K., and Martin, G.R. (1988). Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. U.S.a. 85, 8998–9002.
- Liu, X., and Gorovsky, M.A. (1993). Mapping the 5′ and 3′ ends of Tetrahymena thermophila mRNAs using RNA ligase mediated amplification of cDNA ends (RLM-RACE). Nucleic Acids Res. 21, 4954–4960.
- Ohara, O., Dorit, R.L., and Gilbert, W. (1989). One-sided polymerase chain reaction: the amplification of cDNA. Proc. Natl. Acad. Sci. U.S.A. 86, 5673–5677.
- Scotto Lavino, E., Du, G., and Frohman, M.A. (2007a). 3′ End cDNA amplification using classic RACE. Nat Protoc 1, 2742–2745.
- Scotto Lavino, E., Du, G., and Frohman, M.A. (2007b). 5′ end cDNA amplification using classic RACE. Nat Protoc 1, 2555–2562.
- Upadhyay, A., Moss-Taylor, L., Kim, M.-J., Ghosh, A.C., and O’Connor, M.B. (2017). TGF-β Family Signaling in Drosophila. Cold Spring Harb Perspect Biol 9, a022152.
Transcript
Typically, with novel mRNAs only a portion of its complete sequence is known. The missing nucleotide sequences at the ends of the mRNA can be determined using a PCR-based method called Rapid Amplification of cDNA Ends, or RACE.
In eukaryotes, mature mRNAs have distinctive structural features at both ends. At the five-prime end, most have a methylated guanosine residue connected to the mRNA via a five-prime to five-prime triphosphate linkage. This is also known as the five-prime cap. At the three-prime end, most eukaryotes have a tail of 20 to 250 adenylate residues, called the poly(A) tail.
Now, if the nucleotide sequence of even a small segment is known anywhere within the mRNA, the sequence up to its three-prime end can be amplified using a gene-specific primer and a non-specific oligo(dT) primer that anneals to the poly(A) tail at the three-prime end. This subset of the RACE technique is called three-prime RACE, and it allows for the detection of transcript variants and different three-prime Untranslated Regions, or UTRs.
The sequence at the five-prime end can be amplified similarly. To do this, a poly(A) tail is first attached at the five-prime end. Then, using a non-specific oligo(dT) primer that anneals to the appended tail and a gene-specific primer, the sequence up to the five-prime end can be amplified. This subset of RACE is known as five-prime RACE and is used to find differential five-prime splice variants and alternative five-prime UTRs.
To perform three-prime RACE to identify different transcripts encoded by a given gene, RNA is first isolated from the organism or tissue of interest. Next, cDNA is synthesized from the isolated mRNAs with a reverse transcription reaction that utilizes an oligo(dT) primer and a reverse transcriptase enzyme, which generates complementary DNA from an RNA template. Next, from the generated pool of cDNAs, the target cDNA’s unknown three-prime end is extended via PCR, utilizing the non-specific oligo(dT) primer and a gene-specific primer, and amplified during the PCR reaction.
However, due to the generic nature of the non-specific primer and random mispriming by the gene-specific primer, they can anneal to off-target cDNAs, causing them to amplify as well. To overcome this problem, a second round of PCR is conducted using nested primers, which bind downstream of the first set of primers. This second set of primers further amplifies the cDNA of interest but not the non-specific product, increasing the specificity and yield of the reaction.
Finally, the PCR products are separated using agarose gel electrophoresis, which separates different transcripts of the target gene based on their sizes, producing distinct bands. The band of interest can then be excised, purified, and finally sequenced to obtain the transcript’s complete sequence.
In this video, we will demonstrate the three-prime RACE technique to identify and isolate different transcripts encoded by the Drosophila dSmad2 gene.
Before beginning the synthesis and amplification of the cDNA, use a primer design software to create a five-prime specific primer for the gene of interest, dSmad2 in this example. To ensure the primer is highly specific, it should be around 24 nucleotides and have a Tm ranging from 55 to 65 degrees Celsius. Next, design a second nested primer located three-prime of the first primer, which is also specific for the target sequence.
To begin, extract RNA from 10 whole adult flies with a commercially available RNA extraction kit. Once the RNA is extracted, resuspend the pellet in 20 microliters of nuclease-free water. For a 50-microliter reaction, add 5 microliters of the reaction buffer to each sample tube, and bring up the volume of the reaction by adding 24 microliters of nuclease-free water. Then, add 1 microliter of DNase I to prevent amplification of genomic DNA. Finally, incubate the samples at 37 degrees Celsius for 15 minutes.
After DNase treatment, inactivate the enzyme with 1 microliter of 25 millimolar EDTA in each of the tubes. Add the sample to a spin column to purify the RNA. Next, use a microvolume spectrophotometer to measure the RNA concentration. Adjust the concentration to 100 nanograms per microliter by adding nuclease-free water.
Now, prepare a master mix for the reverse transcription reaction. In addition to the test DNA samples, include one negative control by omitting reverse transcriptase from the appropriate tube. Incubate the reactions for 90 minutes at 42 degrees Celsius. Then, inactivate the reverse transcriptase by incubating the tubes at 85 degrees Celsius for five minutes. Next, dilute the DNA levels by adding 80 microliters of TE buffer to each tube to bring the reaction total to 100 microliters.
Now that the cDNA is synthesized, amplify the gene of interest via PCR. To do this, first prepare the first round amplification PCR mix. In addition to the cDNA template, include a negative control reaction that uses the template from the non-reverse-transcribed product, as well as a reaction that does not have the gene-specific primer as a no-primer control.
Once the reactions are prepared, carry out PCR amplifications in a thermocycler equipped with a heated lid. Next, dilute the products 1 to 20 by adding 1 microliter of the PCR products to 19 microliters of TE buffer. Using these diluted products, prepare the second round amplification PCR mix. Then, use a thermocycler to run the second PCR.
To isolate the PCR fragments, prepare a 1% agarose gel by adding 1 gram of agarose per 100 milliliters of TAE buffer. Melt the mixture for two minutes in the microwave, and then add SYBR Safe stain or ethidium bromide. Pour the molten mixture in a gel tray and allow it to set.
While the gel is setting, pipette 1 microliter droplets of 6X Loading Dye onto a piece of parafilm corresponding to the number of samples. Add 5 microliters of sample to each droplet. Now, load the samples, along with a 1 kilobase DNA ladder, onto the gel. Run the gel at 120 volts for approximately 45 minutes or until the dye front is 75% of the way down the gel.
When the gel has finished running, check the gel bands under a UV transilluminator. Locate the bands and cut them out using a scalpel. Purify the cDNA fragments using a commercially available spin column kit. Once purified, the cDNA fragments can be stored at minus 20 degrees Celsius or used immediately for further analysis.
Prior to this experiment, two transcripts for Drosophila dSmad2 to were annotated in the Drosophila genomic database, FlyBase. Based on the expected splicing of this gene, two products should be identified by the three-prime RACE protocol.
The results from this experiment, however, reveal three different transcripts for dSmad2. Among the predicted products, one transcript is predominant, and one is expressed at a lower level. In addition, a previously undescribed smaller product, visible at 750 base pairs, was detected.