Method for identifying olfactory receptor included in one olfactory cell
View Patent ↗The present invention provides a novel method for identifying an olfactory receptor included in one olfactory cell. In the present invention, amplified is the cDNA derived from the mRNA of the one olfactory cell by a PCR method using a forward primer represented by SEQ ID: 01 and a reverse primer represented by SEQ ID: 02. Subsequently, determined is whether or not a gene sequence of the amplified cDNA corresponds with one gene sequence included in gene sequences coding for olfactory receptors included in the mouse olfactory receptor group A. Finally, determined is that the olfactory receptor included in the one olfactory cell is the olfactory receptor which corresponds to the one gene sequence which corresponds with the gene sequence of the cDNA in the previous step, if the gene sequence of the cDNA corresponds with the one gene sequence in the previous step.
1. A method for identifying an olfactory receptor included in one olfactory cell among a mouse olfactory receptor group A; the method comprising steps of:
(a) obtaining the one olfactory cell;
(b) extracting mRNA contained in the one olfactory cell obtained in the step (a);
(c) obtaining cDNA with reverse transcriptase from the mRNA extracted in the step (b);
(d) amplifying the cDNA obtained in the step (c) by a PCR method using a forward primer represented by SEQ ID: 01 and a reverse primer represented by SEQ ID: 02; wherein
SEQ ID:01 is G (guanine) -C (cytosine) -I (inosine) -T (thymine) -A (adenine) -Y (thymine or cytosine) -G (guanine) -A (adenine) -Y (thymine or cytosine) -C (cytosine) -G (guanine) -I (inosine) -T (thymine) -A (adenine) -Y (thymine or cytosine) -G (guanine) -T (thymine) -I (inosine) -G (guanine) -C (cytosine) -I (inosine) -A (adenine) -T (thymine) -I (inosine) -T (thymine) -G (guanine); and
SEQ ID: 02 is T (thymine) -G (guanine) -Y (thymine or cytosine) -Y (thymine or cytosine) -T (thymine) -G (guanine) -G (guanine) -T (thymine) -I (inosine) -Y (thymine or cytosine) -T (thymine) -I (inosine) -A (adenine) -T (thymine) -I (inosine) -C (cytosine) -Y (thymine or cytosine) -R (guanine or adenine) -T (thymine) -A (adenine) -I (inosine) -A (adenine) -T (thymine) -I (inosine) -A (adenine) -T (thymine) -I (inosine) -G (guanine) -G (guanine) -R (guanine or adenine) -T (thymine) -T (thymine);
(e) determining whether or not a gene sequence of the cDNA amplified in the step (d) corresponds with one gene sequence included in gene sequences coding for olfactory receptors included in the mouse olfactory receptor group A;
wherein the mouse olfactory receptor group A includes the following mouse olfactory receptors:
(Mouse olfactory receptor group A)
Olfr17, Olfr30, Olfr64, Olfr122, Olfr155, Olfr156, Olfr159, Olfr291, Olfr510, Olfr544, Olfr545, Olfr547, Olfr548-ps1, Olfr566, Olfr569, Olfr578, Olfr582, Olfr583, Olfr584, Olfr619, Olfr630, Olfr665, Olfr676, Olfr702, and Olfr713.
2. The method according to claim 1 , further comprising:
disrupting a cell membrane of the one olfactory cell between the step (a) and the step (b).
3. The method according to claim 1 , wherein
the one olfactory cell is extracted from a sample solution containing olfactory cells in the step (a).
4. The method according to claim 1 , wherein
the mouse olfactory receptor group A is selected from the following mouse olfactory receptor group B:
(Mouse olfactory receptor group B)
Olfr17, Olfr64, Olfr122, Olfr156, Olfr291, Olfr510, Olfr547, Olfr548-ps1, Olfr569, Olfr578, Olfr582, Olfr583, Olfr584, Olfr619, Olfr630, Olfr665, Olfr676, and Olfr702.
5. A forward primer represented by SEQ ID: 01 G (guanine) -C (cytosine) -I (inosine) -T (thymine) -A (adenine) -Y (thymine or cytosine) -G (guanine) -A (adenine) -Y (thymine or cytosine) -C (cytosine) -G (guanine) -I (inosine) -T (thymine) -A (adenine) -Y (thymine or cytosine) -G (guanine) -T (thymine) -I (inosine) -G (guanine) -C (cytosine) -I (inosine) -A (adenine) -T (thymine) -I (inosine) -T (thymine) -G (guanine).
6. A reverse primer represented by SEQ ID: 02 T (thymine) -G (guanine) -Y (thymine or cytosine) -Y (thymine or cytosine) -T (thymine) -G (guanine) -G (guanine) -T (thymine) -I (inosine) -Y (thymine or cytosine) -T (thymine) -I (inosine) -A (adenine) -T (thymine) -I (inosine) -C (cytosine) -Y (thymine or cytosine) -R (guanine or adenine) -T (thymine) -A (adenine) -I (inosine) -A (adenine) -T (thymine) -I (inosine) -A (adenine) -T (thymine) -I (inosine) -G (guanine) -G (guanine) -R (guanine or adenine) -T (thymine) -T (thymine).
7. A method of using a forward primer and a reverse primer comprising:
(a) obtaining one olfactory cell;
(b) extracting mRNA contained in the one olfactory cell obtained in the step (a);
(c) obtaining cDNA with reverse transcriptase from the mRNA extracted in the step (b);
(d) amplifying the cDNA obtained in the step (c) by a PCR method using the forward primer and the reverse primer,
wherein the forward primer is represented by SEQ ID: 01 and the reverse primer represented by SEQ ID: 02:
SEQ ID:01 G (guanine) -C (cytosine) -I (inosine) -T (thymine) -A (adenine) -Y (thymine or cytosine) -G (guanine) -A (adenine) -Y (thymine or cytosine) -C (cytosine) -G (guanine) -I (inosine) -T (thymine) -A (adenine) -Y (thymine or cytosine) -G (guanine) -T (thymine) -I (inosine) -G (guanine) -C (cytosine) -I (inosine) -A (adenine) -T (thymine) -I (inosine) -T (thymine) -G (guanine); and
SEQ ID: 02 T (thymine) -G (guanine) -Y (thymine or cytosine) -Y (thymine or cytosine) -T (thymine) -G (guanine) -G (guanine) -T (thymine) -I (inosine) -Y (thymine or cytosine) -T (thymine) -I (inosine) -A (adenine) -T (thymine) -I (inosine) -C (cytosine) -Y (thymine or cytosine) -R (guanine or adenine) -T (thymine) -A (adenine) -I (inosine) -A (adenine) -T (thymine) -I (inosine) -A (adenine) -T (thymine) -I (inosine) -G (guanine) -G (guanine) -R (guanine or adenine) -T (thymine) -T (thymine).
8. The method according to claim 7 further comprising: (e) determining whether or not a gene sequence of the cDNA amplified in the step (d) corresponds with one gene sequence included in a gene sequence coding for a mouse olfactory receptor.