【求助】已知某基因的部分片段，想问下高人能否不做race，直接和基因组序列对比得到其全长！
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本人已知某基因的部分片段，想问下高人能否不做race，直接和基因组序列对比得到其全长！
直接和基因组序列对比得到其全长,是种分析
做到实验时候,还需扩增出来
做到实验时候,还需扩增出来
RACE是一种方法，一般是基因未知的情况下，用此方法得到基因全序列。如果你能通过部分序列进行比对获得全序列，那说明库里已经有全序列在了，那你做此有何意义呢？如果以前没人测过全序列，你知道某部分序列，那可以通过RACE的方法获得全序列，然后递交给NCBI。
附上RACE的方法介绍：
RACE, or Rapid Amplification of cDNA Ends, is a technique used in molecular biology to obtain the full length sequence of an RNA transcript found within a cell. RACE results in the production of a cDNA copy of the RNA sequence of interest, produced through reverse transcription, followed by PCR amplification of the cDNA copies (see RT-PCR). The amplified cDNA copies are then sequenced and, if long enough, should map to a unique mRNA already described, the full sequence of which is known. RACE can provide the sequence of an RNA transcript from a small known sequence within the transcript to the 5' end (5' RACE-PCR) or 3' end (3' RACE-PCR) of the RNA. This technique is sometimes called one-sided PCR or anchored PCR.
The first step in RACE is to use reverse transcription to produce a cDNA copy of a region of the RNA transcript. In this process, an unknown end portion of a transcript is copied using a known sequence from the center of the transcript. The copied region is bounded by the known sequence, and either the 5' or 3' end.
The protocols for 5' or 3' RACES differ slightly. 5' RACE-PCR begins using mRNA as a template for a first round of cDNA synthesis (or reverse transcription) reaction using an anti-sense (reverse) oligonucleotide primer that recognizes a known sequence in the gene of interest; the primer is called a gene specific primer (GSP), and it copies the mRNA template in the 3' to the 5' direction to generate a specific single-stranded cDNA product. Following cDNA synthesis, the enzyme terminal deoxynucleotidyl transferase (TdT) is used to add a string of identical nucleotides, known as a homopolymeric tail, to the 3' end of the cDNA. (There are some other ways to add the 3'-terminal sequence for the first strand of the de novo cDNA synthesised which are much more efficient than homopolymeric tailing, but the sense of the method remains the same). A PCR reaction is then carried out, which uses a second anti-sense gene specific primer (GSP2) that binds to the known sequence, and a sense (forward) universal primer (UP) that binds the homopolymeric tail added to the 3' ends of the cDNAs to amplify a cDNA product from the 5' end.
3' RACE-PCR uses the natural polyA tail that exists at the 3' end of all eukaryotic mRNAs for priming during reverse transcription, so this method does not require the addition of nucleotides by TdT. cDNAs are generated using an Oligo-dT-adaptor primer that complements the polyA stretch and adds a special adaptor sequence to the 5' end of each cDNA. PCR is then used to amplify 3' cDNA from a known region using a sense GSP, and an anti-sense primer complementary to the adaptor sequence
附上RACE的方法介绍：
RACE, or Rapid Amplification of cDNA Ends, is a technique used in molecular biology to obtain the full length sequence of an RNA transcript found within a cell. RACE results in the production of a cDNA copy of the RNA sequence of interest, produced through reverse transcription, followed by PCR amplification of the cDNA copies (see RT-PCR). The amplified cDNA copies are then sequenced and, if long enough, should map to a unique mRNA already described, the full sequence of which is known. RACE can provide the sequence of an RNA transcript from a small known sequence within the transcript to the 5' end (5' RACE-PCR) or 3' end (3' RACE-PCR) of the RNA. This technique is sometimes called one-sided PCR or anchored PCR.
The first step in RACE is to use reverse transcription to produce a cDNA copy of a region of the RNA transcript. In this process, an unknown end portion of a transcript is copied using a known sequence from the center of the transcript. The copied region is bounded by the known sequence, and either the 5' or 3' end.
The protocols for 5' or 3' RACES differ slightly. 5' RACE-PCR begins using mRNA as a template for a first round of cDNA synthesis (or reverse transcription) reaction using an anti-sense (reverse) oligonucleotide primer that recognizes a known sequence in the gene of interest; the primer is called a gene specific primer (GSP), and it copies the mRNA template in the 3' to the 5' direction to generate a specific single-stranded cDNA product. Following cDNA synthesis, the enzyme terminal deoxynucleotidyl transferase (TdT) is used to add a string of identical nucleotides, known as a homopolymeric tail, to the 3' end of the cDNA. (There are some other ways to add the 3'-terminal sequence for the first strand of the de novo cDNA synthesised which are much more efficient than homopolymeric tailing, but the sense of the method remains the same). A PCR reaction is then carried out, which uses a second anti-sense gene specific primer (GSP2) that binds to the known sequence, and a sense (forward) universal primer (UP) that binds the homopolymeric tail added to the 3' ends of the cDNAs to amplify a cDNA product from the 5' end.
3' RACE-PCR uses the natural polyA tail that exists at the 3' end of all eukaryotic mRNAs for priming during reverse transcription, so this method does not require the addition of nucleotides by TdT. cDNAs are generated using an Oligo-dT-adaptor primer that complements the polyA stretch and adds a special adaptor sequence to the 5' end of each cDNA. PCR is then used to amplify 3' cDNA from a known region using a sense GSP, and an anti-sense primer complementary to the adaptor sequence
非常感谢zjubell；是这样的，我们现在做的是家蚕中的某个基因；已经克隆得到了这个基因的部分cDNA序列（通过收索相近物种得到同源保守序列，ncbi上收索EST并拼接，然后设计引物）；一般来说是要通过race的方法来向两端拉一下得到全长。但是我们初步想着可不可以用这段已经p出来的序列去和已公布的家蚕基因组序列去比对，确定其在基因组上的位置。然后再向两边分析得到这个基因的全长序列，同时也把内含子，外显子和表达调控元件分析出来。这种思路是不是存在什么问题，是否可行，还请高人不吝赐教！
heroyl wrote:
非常感谢zjubell；是这样的，我们现在做的是家蚕中的某个基因；已经克隆得到了这个基因的部分cDNA序列（通过收索相近物种得到同源保守序列，ncbi上收索EST并拼接，然后设计引物）；一般来说是要通过race的方法来向两端拉一下得到全长。但是我们初步想着可不可以用这段已经p出来的序列去和已公布的家蚕基因组序列去比对，确定其在基因组上的位置。然后再向两边分析得到这个基因的全长序列，同时也把内含子，外显子和表达调控元件分析出来。这种思路是不是存在什么问题，是否可行，还请高人不吝赐教！
跟已知物种去比对，然后确定在基因组上的大致位置是可行的。
再向两边分析得到全长，这个也许不行，毕竟两个物种肯定会有差异，要得到全长序列，唯一金标准就是测序，所以你向两边分析，得到‘可能’的序列，再用此序列设计引物，做PCR，再测序，这样才能确定全长。
至于把内含子，外显子和表达元件，也许存在难度。首先，内含子一般比较长的吧(人的内含子，一般都是几十Kb，甚至几百Kb的），光PCR可能困难（当然也有可能你那个基因很短，很容易弄）。而外显子，如果你光通过基因组也许还不行，最好通过mRNA逆转，再测序与基因组对比，就很容易找到外显子序列了。
你的总体思路应该没啥大问题，但必须case by case
非常感谢！！
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