Identification of ion channel

1
Identification of ion channel

• How to detect any particular mRNA in any given
  cell?

2
How to identify individual channels expressed in
a given cell?
Antibody? Pharmacological compound?
RT-PCR
3
The most important component of RT-PCR 2
specific primers for targeted mRNA
alternatively possible 1 specific gene primer
and 1 of sequence found in gene family

4
How to obtain gene specific Primers?????
Several possibilities
5
Commercially available ? Published primer
sequence
Before ordering at least verify that they will
recognize mRNA of interest of the correct specie
http//www.ncbi.nlm.nih.gov/BLAST/
Nucleotide .Quickly search for highly similar
sequences (megablast) .Quickly search for
divergent sequences (discontiguous megablast)
.Nucleotide-nucleotide BLAST (blastn)
.Search for short, nearly exact matches
.Search trace archives with megablast or
discontiguous megablast
6
Design your own primer!
Several software available PC oligo6
Enter nucleotide sequence, program select forward
and reverse primers Provides annealing
temperature and length of PCR product Selects
sequences without secondary structures
7
WEB based program
http//frodo.wi.mit.edu/cgi-bin/primer3/primer3_ww
w.cgi
8
Get to know the molecule you are trying to
detect!
How?
http//www.ncbi.nlm.nih.gov/Database/index.html

• Pubmed
• Protein
• nucleotide

Search for conserved domain in protein sequence
http//www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.
cgi
9
Swissprot protein database entries include
informations like
Transmenbrane domain Ankyrin repeats Ion channel
Catalytic domain Etc
Conserved domains
http//au.expasy.org/sprot/
10
Additional features are searchable
Potential protein-protein interaction
domains ankyrin repeat domain obtainable
from swissprot entry coil-coiled domain are
searchable with different programs
http//www.ch.embnet.org/software/COILS_form.html
11
Trpc1 human
12
Other program available from same site
http//www.isrec.isb-sib.ch/webmarcoil/webmarcoilC
1.html
Precise sequence and position of coiled-coil
13
Marcoil1.0      (C)Mauro Delorenzi Prediction
of coiled-coil domains in protein sequences by
posterior probabilities generated by a hidden
markov model MARCOIL-PAGE Go to the information
page Submissions with Internet Explorer can give
problems. Please let me know Mauro.Delorenzi_at_isre
c.unil.ch --------------------------------------
---------------------------------- JOBNAME
(optional) PARAMETERS (optional) SEQUENCE
(max length 11000 aa per protein)
1. Coiled-Coil Emission P. Matrix Paste
sequences in (multi)fasta format or.
2. HMM Transition P. Matrix.
Either precomputed (2a, default) or user-defined
use the file upload (2b) 2a.
Precomputed ("gtseqname" between sequences) 2b.
User-defined Take the values entered
below Parameter   i  (default0.0001) 
Parameter   t  (default0.01)  Or upload
sequence data file (no special characters in the
address!)   Parameter   r  (default 0.00001) 
SUBMIT    CLEAR ---------------------------------
---------------------------------------   

Protein sequence
14
MARCOILS PREDICTIONS 1. SUMMARY OF COILED-COIL
DOMAINS AT DIFFERENT LEVELS OF STRINGENCY
PREDICTED DOMAINS AT THRESHOLD 1 1. from 33
to 49 (length 17) with max 2.4 2. from 196
to 290 (length 95) with max 73.4 3. from
413 to 425 (length 13) with max 1.6 4. from
678 to 699 (length 22) with max 2.6 5. from
705 to 750 (length 46) with max 89.5
PREDICTED DOMAINS AT THRESHOLD 10 1. from
202 to 258 (length 57) with max 73.4 2.
from 708 to 746 (length 39) with max 89.5
PREDICTED DOMAINS AT THRESHOLD 50 1. from
205 to 249 (length 45) with max 73.4 2.
from 715 to 744 (length 30) with max 89.5
PREDICTED DOMAINS AT THRESHOLD 90
PREDICTED DOMAINS AT THRESHOLD 99
15
Comprehensive search for specific domains in
protein sequence (except protein secondary
structure, coiled-coil, helix, etc) Include
phosphorylation, glycosylation
http//myhits.isb-sib.ch/cgi-bin/motif_scan
16
  Motif Scan user anonymous log in Protein
Sequence Input Enter a protein sequence in RAW or
FASTA or Swiss-Prot format or a dbAC or dbID
identifier Clear input Reset page Motif scanning
means finding all known motifs that occur in a
sequence. This form lets you paste a protein
sequence, select the collections of motifs to
scan for, and launch the search. Some general
documentation is available about the Prosite and
Pfam collections of motifs. Another document
deals with the interpretation of the match
scores. You should consult the home pages of
Prosite on ExPASy, Pfam and InterPro for
additional information. Warning The scan might
take a few minutes, thus if your proteins of
interest are already in the sequence databases
(see list), the Query by Protein form is much
faster, and the Protein Hub provides a collection
of tools that you might find useful. Parameters
Database of motifs (db description) PROSITE
patterns PROSITE patterns (frequent match
producers) PROSITE profiles Prefile (more
profiles) Na-channel profiles HAMAP profiles Pfam
HMMs (local models) Pfam HMMs (global
models) search
17
Additional program can be used to identify all
potentially phosphorylated residues
http//www.cbs.dtu.dk/services/NetPhos/
18
More/different protein features on central EMBL
web site
http//www.ebi.ac.uk/interpro/

•   Home
• InterPro is a database of protein families,
  domains and functional sites in which
  identifiable features found in known proteins can
  be
• applied to unknown protein sequences.
• Further information on InterPro can be found in
  the documentation - see links on the left hand
  side.
• For information, comments and/or suggestions on
  the InterPro database, please contact us at EBI
  Support.
• Search
• Search - help - example kinase
• Search InterPro
• Updated Documents and New Links
• Announcement
• InterPro release 11.0 is out with increased
  coverage and functionality.
• News
• Release 11.0 contains 12294 entries and covers
  77.5 of UniProt - 1620653 of 2090735 proteins

keyword
19
TRPC1 calcium channel
http//www.ebi.ac.uk/interpro/DisplayIproEntry?ac
IPR005457
20
  ClustalW Valid format for input is
FASTA(Pearson) max number of sequences 30 max
total length of sequences 10000 Help
page ---------------------------------------------
--------------------------- Scoring matrix
    Opening gap penalty 10 Extending gap
penalty 0.05 End gap penalty
10 Separation gap penalty 0.05 Output format
Clustal Output order inpuy Input
sequences (see above for valid
formats) Run ClustalW Clear Input
Blosum
gthuman MM gtrat M. gtchicken M..
21
ClustalW query receipt ---------------------------
--------------------------------------------- Than
ks. Your request has been filed with the
following data Number 10079 e-mail
Format aln Processing, please
wait... Here are your search results Multiple
alignments Dendrograms clustalw (aln)
clustalw (dnd) GCG/MSF PIR GDE phylip Thank
you for using ClustalW
22
Partial clustal alignment
human ATGATGGCGGCCCTGTACCCGAGCACGGACCTCT
CGGGCGCCTCCTCCTCCTCCCTGCCT rat
ATGATGGCGGCCCTGTACCCGAGCACGGACCTCTCTGGCGTCTCCTCCTC
CTCCCTGCCT chicken ---ATGGCGGCTCTGTACCAGAG
CGCGGACCCGTCCG-----------------------

human
TCCTCTCCATCCTCTTCCTCGCCGAACGAGGTGATGGCGCTGAAGGATGT
GCGGGAGGTG rat TCCTCCCCATCCTCCTCATCGCC
CAACGAAGTGATGGCGCTGAAGGATGTGCGAGAGGTG chicken
----------CCTCGGCCTCGCCCAACAAGCTGCTGGCGCTGAAGG
ATGTGCGGCAGGTG
human
AAGGAGGAGAATACGCTGAATGAGAAGCTTTTCTTGCTGG
CGTGCGACAAGGGTGACTAT rat
AAGGAGGAGAACACCTTGAATGAGAAGCTTTTCTTGCTGGCGTGCGACAA
GGGTGACTAT chicken AAGGAAGAGACCACACTGGACGA
GAAGCTTTTCCTGCTGGCCTGTGACAAAGGTGATTAT

human
TATATGGTTAAAAAGATTTTGGAGGAAAACAGTTCAGGTGACTTGAACAT
AAATTGCGTA rat TATATGGTTAAAAAGATTTTGGA
GGAAAACAGTTCAGGTGACTTGAACATAAATTGCGTA chicken
TACATGGTTAAGAAACTCTTAGAAGAAAACAGCTCGGGTGAGATGA
ACATAAACTGCGTG
human
GATGTGCTTGGGAGAAATGCTGTTACCATAACTATTGAAA
ACGAAAACTTGGATATACTG rat
GATGTGCTTGGGAGAAATGCTGTTACCATAACTATTGAAAACGAAAGCTT
GGATATACTG chicken GATGTGCTTGGAAGAAATGCTGT
TACCATAACCATTGAAAATGAAAATTTGGACATACTA

human
CAGCTTCTTTTGGACTACGGTTGTCAG-----------------------
---------- rat CAGCTGCTTTTGGACTACGGTTG
TCAG--------------------------------- chicken
CAGCTTCTTCTGGATTATGGGTGCCAGTCATCAGATGCACTTTTGG
TGGCTATTGACTCA
human
----------------------------------------
-------------------- rat
--------------------------------------------------
---------- chicken GAAGTGGTGGGAGCTGTTGACAT
TCTACTTAATCACCGACCGAAAAGATCCTCCAGGCCA

human
---------AAACTAATGGAACGAATTCAGAATCCTGAGTATTCAACAAC
TATGGATGTT rat ---------AAACTTATGGAACG
AATTCAGAATCCGGAATACTCAACAACAATGGATGTT chicken
ACCATTGTAAAACTAATGGAACGCATTCAGAACCCAGAATACTCAA
CAACCATGGATGTG

human GCACCTGTCATTTTAGCTGCTCATCGTAACAA
CTATGAAATTCTTACAATGCTCTTAAAA rat
GCACCAGTTATTTTAGCTGCTCATCGTAACAACTATGAGATCCTCACAAT
GCTTCTGAAG chicken GCACCTGTCATCTTAGCTGCTCA
TCGTAATAACTATGAAATTCTCACCATGCTGTTAAAG


Can this region be used to design trpc1 specific
primers?
23
Clustal w trpc1 aa 564 to 617
humantrpc1 --------------------TCTCCTTAG
CGCATGTGGCAATCTTTGTCACAAGATTTAG mousetrpc1b
TGCTCTGTTCTGGTACATCTTCTCCTTAGCGCACGTGGCCATCTTTGTCA
CCAGGTTTAG trpc1cow -----------------TTTTC
TCCTTAGCACATGTGGCAATCTTTGTCACAAGATTTAG

humantrpc1
CTATGGAGAAGAACTGCAGTCCTTTGTGGGAGCTGTCATTGTTGGTACAT
ACAATGTCGT mousetrpc1b CTATGGGGAAGAGCTGCAGTCC
TTCGTTGGAGCTGTGATTGTTGGAACTTACAATGTTGT trpc1cow
CTACGGAGAAGAATTACAGTCCTTCGTCGGAGCTGTTATTGTTG
GGACATACAACGTTGT

humantrpc1 GGTTGTGATTGTGCTTACCAAACTGCTGG
TGGCAATGCTTCATAAAAGCTTTCAGTTGA- mousetrpc1b
GGTTGTGATTGTGCTGACGAAGCTGCTCGTGGCGATGCTTCACAAGAGCT
TCCAGCTGAT trpc1cow GGTTGTGATAGTACTTACCAAG
CTGCTGGTGGCAATGCTTCATAAAAGTTTTCAGTTGAT


24
Sequences producing significant alignments
(Bits) Value gi27545448refN
M_003304.3 Homo sapiens transient receptor ...
196 4e-48 gi55621247refXM_516794.1
PREDICTED Pan troglodytes trans... 196
4e-48 gi1359889embZ73903.1HSTRPC1A
H.sapiens mRNA for TRPC1A 196 4e-48
gi23307962gbAC022291.27 Homo sapiens 3 BAC
RP11-71O21 (Ro... 196 4e-48 gi1370118embX8
9066.1HSTRPC1GN H.sapiens mRNA for TRPC1 prote
196 4e-48 gi1072042gbU31110.1HSU31110
Human alternatively spliced t... 196 4e-48
gi50429190gbAY572429.1 Cavia porcellus
transient receptor... 143 5e-32 gi15082129g
bAF395783.1AF395783 Cavia porcellus TRP1 mRNA,
pa 135 1e-29 gi5759113gbAF170493.1AF17049
3 Oryctolagus cuniculus putat... 135
1e-29 gi17646729gbAF448492.1 Cricetulus
griseus transient recep... 125
1e-26 gi73697871gbDQ151456.1 Bos taurus
transient receptor pote... 121
2e-25 gi73697869gbDQ151455.1 Bos taurus
transient receptor pote... 121
2e-25 gi73697867gbDQ151454.1 Bos taurus
transient receptor pote... 121 2e-25
gi73990664refXM_542818.2 PREDICTED Canis
familiaris simi... 117 3e-24
gi25742644refNM_053558.1 Rattus norvegicus
transient rece... 109 8e-22
gi62654132refXM_579562.1 PREDICTED Rattus
norvegicus hyp... 109 8e-22
gi3786395gbAF061266.1AF061266 Rattus
norvegicus trp1 beta va 109 8e-22
gi3139061gbAF061873.1AF061873 Rattus
norvegicus transient... 109 8e-22
gi6755884refNM_011643.1 Mus musculus
transient receptor p... 103 5e-20
gi14389317gbAC091531.9 Mus musculus Strain
C57BL6/J Chrom... 103 5e-20 gi62177099gbAC
157947.2 Mus musculus BAC clone RP23-238L21...
103 5e-20 gi6179957gbAF191551.1AF191551
Mus musculus putative Ca in... 103 5e-20
gi2052506gbU95169.1MMU95169 Mus musculus
Mtrp1 delta variant 103 5e-20
gi2052505gbU95168.1MMU95168 Mus musculus
Mtrp1 gamma variant 103 5e-20
gi2052503gbU95167.1MMU95167 Mus musculus
Mtrp1 beta variant 103 5e-20
gi1373058gbU40980.1MMU40980 Mus musculus
trp-related protein 103 5e-20
gi1825486gbU73625.1MMU73625 Mus musculus
Mtrp1 alpha (Mtrp1) 103 5e-20
gi31341889refNM_174476.2 Bos taurus
transient receptor poten 89.7 7e-16
gi2331276gbAF012901.1AF012901 Bos taurus
plasmalemma cation 89.7 7e-16
gi2331274gbAF012900.1AF012900 Bos taurus
plasmalemma cation 89.7 7e-16
25
gi34365780refNM_183354.1 Mus musculus solute
carrier fami... 38.2 2.3
gi6755531refNM_011389.1 Mus musculus solute
carrier famil... 38.2 2.3
gi48675555gbAC140447.3 Mus musculus BAC
clone RP23-53E23 ... 38.2 2.3
gi16877250gbBC016888.1 Mus musculus solute
carrier family... 38.2 2.3
gi21322405embAL732389.4 Human DNA sequence
from clone RP1... 38.2 2.3
gi30047891gbBC051100.1 Mus musculus solute
carrier family... 38.2 2.3
gi22657473gbAC129803.3 Homo sapiens 3 BAC
RP11-15N24 (Ros... 38.2 2.3
gi21070776gbAC115621.3 Homo sapiens BAC
clone RP11-315D13 fr 38.2 2.3
gi6604531gbAC009405.3AC009405 Homo sapiens
BAC clone RP11-17 38.2 2.3
gi17425248dbjAP003025.2 Homo sapiens
genomic DNA, chromos... 38.2 2.3
gi4097436gbU61381.1MMU61381 Mus musculus
apical bumetanid... 38.2 2.3
gi28874631embAL929451.8 Mouse DNA sequence
from clone RP2... 38.2 2.3
gi1079520gbU20975.1MMU20975 Mus musculus
kidney-specific ... 38.2 2.3
gi1079516gbU20973.1MMU20973 Mus musculus
kidney-specific ... 38.2 2.3
gi55831570gbAC131693.5 Mus musculus BAC
clone RP23-391D10 fr 38.2 2.3
gi2290525gbU94518.1MMU94518 Mus musculus
strain CD1 Na-(K... 38.2 2.3
gi1079518gbU20974.1MMU20974 Mus musculus
kidney-specific ... 38.2 2.3
gi55734044gbAC120841.7 Mus musculus
chromosome 8, clone RP24 36.2 9.2
gi74039677gbAC164634.3 Mus musculus BAC
clone RP23-64E16 ... 36.2 9.2
gi15130744embAL590486.9 Human DNA sequence
from clone RP1... 36.2 9.2
gi10120085embAL359894.9 Human DNA sequence
from clone RP1... 36.2 9.2
gi33086431gbAC124530.4 Mus musculus BAC
clone RP23-286D7 ... 36.2 9.2
gi34495051gbAC117232.3 Mus musculus BAC
clone RP23-405K2 ... 36.2 9.2
gi27413949gbAC122055.4 Mus musculus BAC
clone RP24-465O15... 36.2 9.2
gi24476143gbAC122384.3 Mus musculus BAC
clone RP24-63P8 f... 36.2 9.2
gi22213512gbAC121600.2 Mus musculus BAC
clone RP23-302K1 fro 36.2 9.2
gi34368610gbAC103574.7 Rattus norvegicus 1
BAC CH230-205C... 36.2 9.2
gi18958586gbAC055753.21 Homo sapiens 3 BAC
RP11-503I22 (R... 36.2 9.2
gi22830385gbAC103688.4 Homo sapiens
chromosome 8, clone CTD- 36.2 9.2
gi22539130gbAC104976.3 Homo sapiens
chromosome 8, clone RP11 36.2 9.2
gi21217399gbAC009244.25 Homo sapiens
chromosome 7 clone RP11 36.2 9.2
gi20128341gbAC011124.9 Homo sapiens
chromosome 8, clone RP11 36.2 9.2
gi67625941gbAC159097.5 Mus musculus
chromosome 8, clone RP24 36.2 9.2
gi18644827gbAC083934.3 Homo sapiens
chromosome 8, clone RP11 36.2 9.2
gi16799064gbAC096770.3 Homo sapiens BAC
clone RP11-755P13 fr 36.2 9.2
gi62945436gbAC153559.4 Mus musculus 10 BAC
RP23-86P1 (Ros... 36.2 9.2
gi62000951gbAC147282.3 Pan troglodytes BAC
clone RP43-86L... 36.2 9.2
gi57470743gbAC132472.3 Mus musculus BAC
clone RP23-113B13... 36.2 9.2
gi32166006embAL772280.10 Mouse DNA sequence
from clone RP... 36.2 9.2 gi51921479gbAC1
24772.4 Mus musculus BAC clone RP23-47P3 from
36.2 9.2 gi57012504gbAC122351.5 Mus
musculus BAC clone RP23-388H1 fro 36.2 9.2
26
gtgi55621247refXM_516794.1 PREDICTED Pan
troglodytes transient receptor potential cation
channel, subfamily C, member 1
(LOC460747), mRNA Length4339 Score
196 bits (99), Expect 4e-48 Identities
99/99 (100), Gaps 0/99 (0)
StrandPlus/Plus Query 1 CTCCTTAGCGCATGTGGCA
ATCTTTGTCACAAGATTTAGCTATGGAGAAGAACTGCAGTC 60

Sbjct 2112
CTCCTTAGCGCATGTGGCAATCTTTGTCACAAGATTTAGCTATGGAGAAG
AACTGCAGTC 2171 Query 61 CTTTGTGGGAGCTGTCATT
GTTGGTACATACAATGTCGT 99
Sbjct
2172 CTTTGTGGGAGCTGTCATTGTTGGTACATACAATGTCGT
2210 gtgi17646729gbAF448492.1 Cricetulus
griseus transient receptor potential-like protein
(TRP1) mRNA, partial cds
Length428 Score 125 bits (63), Expect
1e-26 Identities 90/99 (90), Gaps 0/99
(0) StrandPlus/Plus Query 1
CTCCTTAGCGCATGTGGCAATCTTTGTCACAAGATTTAGCTATGGAGAAG
AACTGCAGTC 60
Sbjct
230 CTCCTTAGCACATGTGGCCATCTTTGTCACAAGGTTTAGCTATGG
AGAAGAACTGCAGTC 289 Query 61
CTTTGTGGGAGCTGTCATTGTTGGTACATACAATGTCGT 99

Sbjct 290 CTTCGTTGGAGCTGTGATTGTTGGGACGTACA
ACGTCGT 328 gtgi34365780refNM_183354.1 Mus
musculus solute carrier family 12, member 1
(Slc12a1), transcript variant 2,
mRNA Length4655 Score 38.2 bits
(19), Expect 2.3 Identities 19/19 (100),
Gaps 0/19 (0) StrandPlus/Minus Query 47
GAAGAACTGCAGTCCTTTG 65
Sbjct 1820
GAAGAACTGCAGTCCTTTG 1802
27
One important precaution Make sure that your
PCR product contains exon-exon boundaries to
avoid amplification of nuclear unspliced RNA or
DNA
28
Can our blast search tell us if our selected PCR
product is a good choice?
 Display  Show  All 1  Genes Genomes
1  SNP GeneView 1  1 TRPC1 Links Official
Symbol TRPC1 and Name transient receptor
potential cation channel, subfamily C, member 1
Homo sapiens Other Aliases HGNC12333, HTRP-1,
TRP1 Other Designations transient receptor
potential channel 1 Chromosome 3 Location
3q22-q24 GeneID 7220
gtgi23307962gbAC022291.27 Homo sapiens 3
BAC RP11-71O21 (Roswell Park Cancer Institute
Human BAC Library) complete sequence
Length28478 Score 196 bits (99),
Expect 4e-48 Identities 99/99 (100), Gaps
0/99 (0) StrandPlus/Plus Query 1
CTCCTTAGCGCATGTGGCAATCTTTGTCACAAGATTTAGCTATGGAGAAG
AACTGCAGTC 60
Sbjct
14905 CTCCTTAGCGCATGTGGCAATCTTTGTCACAAGATTTAGCTAT
GGAGAAGAACTGCAGTC 14964 Query 61
CTTTGTGGGAGCTGTCATTGTTGGTACATACAATGTCGT 99
Sb
jct 14965 CTTTGTGGGAGCTGTCATTGTTGGTACATACAATGTCG
T 15003
29
We need to increase the size of PCR product to
include intron(s)
If we look in detail at the alignment it also
appears that a 19 nucleotides stretch is also
homologous to another mRNA
gtgi16877250gbBC016888.1 Mus musculus solute
carrier family 12, member 1, transcript variant
2, mRNA (cDNA clone MGC18410
IMAGE4236020), complete cds
Length4650 Score 38.2 bits (19), Expect
2.3 Identities 19/19 (100), Gaps 0/19 (0)
StrandPlus/Minus Query 47
GAAGAACTGCAGTCCTTTG 65
Sbjct 1800
GAAGAACTGCAGTCCTTTG 1782
30
WEB based program
http//frodo.wi.mit.edu/cgi-bin/primer3/primer3_ww
w.cgi
31
(No Transcript)
32
-Cut paste sequence of interest trpm8 rat
-selected region of interest 700, 4
Primer around 4 base in position 701,2,3,4
-select annealing temperature and other desired
features of the primers
33
(No Transcript)
34
Primer3 Output -----------------------------------
------------------------------------- WARNING
Numbers in input sequence were deleted. PRIMER
PICKING RESULTS FOR trpm8 No mispriming library
specified Using 1-based sequence positions OLIGO
start len tm gc any 3'
seq LEFT PRIMER 670 20 57.91 50.00
6.00 2.00 gacaccctcatcaggaattg RIGHT PRIMER
892 20 58.02 45.00 4.00 2.00
tgccaccatagttggaatct SEQUENCE SIZE 3315 INCLUDED
REGION SIZE 3315 PRODUCT SIZE 223, PAIR ANY
COMPL 3.00, PAIR 3' COMPL 1.00 TARGETS (start,
len) 700,4
661
tccaaccgggacaccctcatcaggaattgcgatgctgagggctatttttt
agcccagtac gtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgt
721
cttatggatgacttcacaagagatccactgtatatcctggacaacaacca
cacacatttg
781
ctgctcgtggacaatggctgtcatggacatcccactgtcgaagcaaagct
ccggaatcag
841
ctagagaagtatatctctgagcgcactattcaagattccaactatggtgg
caagatcccc
ltltltltltltltltltltltltltltltltltltltlt
35
KEYS (in order of precedence)
target gtgtgtgtgtgt left primer ltltltltltlt right
primer ADDITIONAL OLIGOS
start len tm gc any 3' seq 1
LEFT PRIMER 618 20 57.89 45.00 6.00
3.00 ggagaatattgtggccattg RIGHT PRIMER
783 20 58.01 45.00 4.00 0.00
cagcaaatgtgtgtggttgt PRODUCT SIZE 166, PAIR
ANY COMPL 4.00, PAIR 3' COMPL 0.00 2 LEFT
PRIMER 602 20 58.25 55.00 3.00
0.00 tcagcaggagttcagaggag RIGHT PRIMER
783 20 58.01 45.00 4.00 0.00
cagcaaatgtgtgtggttgt PRODUCT SIZE 182, PAIR
ANY COMPL 2.00, PAIR 3' COMPL 0.00 3 LEFT
PRIMER 603 20 58.25 55.00 3.00
0.00 cagcaggagttcagaggaga RIGHT PRIMER
783 20 58.01 45.00 4.00 0.00
cagcaaatgtgtgtggttgt PRODUCT SIZE 181, PAIR
ANY COMPL 2.00, PAIR 3' COMPL 1.00 4 LEFT
PRIMER 595 20 57.71 50.00 5.00
2.00 aacaccatcagcaggagttc RIGHT PRIMER
783 20 58.01 45.00 4.00 0.00
cagcaaatgtgtgtggttgt PRODUCT SIZE 189, PAIR
ANY COMPL 5.00, PAIR 3' COMPL 2.00 Statistics
con too in in no tm
tm high high high sid
many tar excl bad GC too too any
3' poly end ered Ns get
reg GC clamp low high compl compl X
stab ok Left 6167 0 0 0 59
0 819 4358 0 1 0 73
857 Right 23102 0 0 0 54 0
4397 14189 0 3 47 300 4112 Pair
Stats considered 2995, unacceptable product size
2989, ok 6 primer3 release 1.0 (primer3_www_resu
lts.cgi v 0.4)
36
What should I do now?
37
Sequence to be checked with blast
tccaaccgggacaccctcatcaggaattgcgatgctgagggctatttttt
agcccagtac cttatggatgacttcacaagagatccactgtatatcctg
gacaacaaccacacacatttg ctgctcgtggacaatggctgtcatggac
atcccactgtcgaagcaaagctccggaatcag ctagagaagtatatctc
tgagcgcactattcaagattccaactatggtggcaagat
Better to select mammals instead of entire
database
38
gtgi21361690refNM_024080.3 Homo sapiens
transient receptor potential cation channel,
subfamily M, member 8 (TRPM8), mRNA
Length5674 Score 468 bits (236), Expect
2e-129 Identities 236/236 (100), Gaps 0/236
(0) StrandPlus/Plus Query 1
TCCAACCGGGACACCCTCATCAGGAATTGCGATGCTGAGGGCTATTTTTT
AGCCCAGTAC 60
Sbjct 701
TCCAACCGGGACACCCTCATCAGGAATTGCGATGCTGAGGGCTATTTTTT
AGCCCAGTAC 760 Query 61 CTTATGGATGACTTCACAAGA
GATCCACTGTATATCCTGGACAACAACCACACACATTTG 120

Sbjct 761 CTTATGGATGACTTCACAAG
AGATCCACTGTATATCCTGGACAACAACCACACACATTTG
820 Query 121 CTGCTCGTGGACAATGGCTGTCATGGACATCCC
ACTGTCGAAGCAAAGCTCCGGAATCAG 180

Sbjct 821 CTGCTCGTGGACAATGGCTGTCATGGA
CATCCCACTGTCGAAGCAAAGCTCCGGAATCAG 880 Query
181 CTAGAGAAGTATATCTCTGAGCGCACTATTCAAGATTCCAACTAT
GGTGGCAAGAT 236
Sbjct 881
CTAGAGAAGTATATCTCTGAGCGCACTATTCAAGATTCCAACTATGGTGG
CAAGAT 936
39
1 TRPM8 Links Official Symbol TRPM8 and Name
transient receptor potential cation channel,
subfamily M, member 8 Homo sapiens Other
Aliases HGNC17961, LTRPC6, MGC2849, TRPP8 Other
Designations short form of the TRPM8 cationic
channel transient receptor potential subfamily M
member 8 Chromosome 2 Location 2q37.1 GeneID
79054
40
gi45774091gbAC005538.3 Homo sapiens BAC
clone RP11-332L11 from 2, complete sequence
Length193627 Score 351 bits (177),
Expect 3e-94 Identities 177/177 (100), Gaps
0/177 (0) StrandPlus/Plus Query 38
AGGGCTATTTTTTAGCCCAGTACCTTATGGATGACTTCACAAGAGATCCA
CTGTATATCC 97
Sbjct
119610 AGGGCTATTTTTTAGCCCAGTACCTTATGGATGACTTCACAA
GAGATCCACTGTATATCC 119669 Query 98
TGGACAACAACCACACACATTTGCTGCTCGTGGACAATGGCTGTCATGGA
CATCCCACTG 157
Sbjct
119670 TGGACAACAACCACACACATTTGCTGCTCGTGGACAATGGCT
GTCATGGACATCCCACTG 119729 Query 158
TCGAAGCAAAGCTCCGGAATCAGCTAGAGAAGTATATCTCTGAGCGCACT
ATTCAAG 214
Sbjct 119730
TCGAAGCAAAGCTCCGGAATCAGCTAGAGAAGTATATCTCTGAGCGCAC
TATTCAAG 119786 Score 79.8 bits (40),
Expect 2e-12 Identities 40/40 (100), Gaps
0/40 (0) StrandPlus/Plus Query 1
TCCAACCGGGACACCCTCATCAGGAATTGCGATGCTGAGG 40

Sbjct 116466 TCCAACCGGGACACCCTCATCAGGAATTGCGATG
CTGAGG 116505 Score 48.1 bits (24), Expect
0.007 Identities 24/24 (100), Gaps 0/24
(0) StrandPlus/Plus Query 213
AGATTCCAACTATGGTGGCAAGAT 236
Sbjct 120842
AGATTCCAACTATGGTGGCAAGAT 120865
There is an intron here!
41
gtgi42742435gbAY532376.1 Homo sapiens short
transient receptor potential subfamily M member
8, variant beta (TRPM8) mRNA, complete
cds, alternatively spliced
Length768 Score 79.8 bits (40), Expect
2e-12 Identities 40/40 (100), Gaps 0/40
(0) StrandPlus/Plus Query 1
TCCAACCGGGACACCCTCATCAGGAATTGCGATGCTGAGG 40
Sb
jct 599 TCCAACCGGGACACCCTCATCAGGAATTGCGATGCTGAGG
638 gtgi42742433gbAY532375.1 Homo sapiens
short transient receptor potential subfamily M
member 8, variant alpha (TRPM8)
mRNA, complete cds, alternatively
spliced Length722 Score 79.8 bits
(40), Expect 2e-12 Identities 40/40 (100),
Gaps 0/40 (0) StrandPlus/Plus Query 1
TCCAACCGGGACACCCTCATCAGGAATTGCGATGCTGAGG 40
Sb
jct 553 TCCAACCGGGACACCCTCATCAGGAATTGCGATGCTGAGG
592
42
Significance for our primer design?
We should go back to our analysis and determine
what our aims are before we decide whether we
should use these primers
43
Check primers
http//www.ncbi.nlm.nih.gov/BLAST/
Nucleotide
Search for short, nearly exact matches
After entering primer sequences select specy homo
sapiens To try to pick up as many sequences as
possible
44
gtgi14041750embAL359208.14 Human DNA
sequence from clone RP11-320N6 on chromosome 13,
complete sequence
Length139649 Score 38.2 bits (19), Expect
0.30 Identities 22/23 (95), Gaps 0/23 (0)
StrandPlus/Minus Query 12
CAGGAATTGTGCCACCATAGTTG 34
Sbjct 87748
CAGGAATTGTGCCACCATGGTTG 87726 Score 24.3
bits (12), Expect 4532 Identities 15/16
(93), Gaps 0/16 (0) StrandPlus/Plus Query
4 ACCCTCATCAGGAATT 19
Sbjct 45216 ACCCTCATCAGTAATT
45231
45
What happens now?
Select commercial suppliers and order price
should be around 10 for an 18 base oligo
46
Setting up and optimizing
Without gradient it is safer to start at lowest
temperature Around 10oC below Tm (melting
temperature)
47
How many cycles?
Starting material approximately 5 ug total RNA
1-3 mRNA (50-150ng) cDNA synthesis 90-100
(when using RNAseH minus reverse
transcriptase) When using 2.5 ng to 7.5 ng of
mRNA most actively transcribed mRNA should be
detectable with 30 to 35 cycles
So does it really work?
48
But first the PCR product must be the right size!
The best way to know is to sequence the PCR
product
It is possible to use restriction enzyme to check
whether restriction pattern is compatible with
expected PCR product
It is possible to use third (and a fourth) primer
recognizes internal sequence and will produce
shorter PCR product
How I can I know?
49
trpc 1
Nb2A mouse
HEK SHSY5Y IMR32
trpc1 trpc 3 trpc 6
500
trpc7
Pc12 rat
trpc6
500
50
The pictures showed RT-PCR from commonly used
organism (rat, mouse, human) for which sequences
are available.
But it will not always work 1 set of conditions
that works for one type of human cells may not
work so well in another
It is possible to detect dramatic changes in mRNA
level but quantification is not really possible
requires real-time PCR
51
Quantification (old-fashioned/difficult/ time-cons
uming but does not require specialized apparatus)
52
When ones needs primers for sequences not in the
database, like for CHO cells it is more difficult
and time consuming and amplified region must be
chosen carefully so identification is 100
53
CLUSTAL W (1.8) multiple sequence alignment

trpm2cho
-------AGTGATTATGATGAAGGATGTCTTCTTCTTCCTCTTTCTCCTG
GCGGTATGGG trpm2hsfr --------------ATGATGAAG
GACGTCTTCTTCTTCCTCTTCCTGCTGGCTGTGTGGG trpm2rat
-------AGTGATTATGATGAAGGATGTCTTCTTCTTCCTCTTTCT
CCTGGCGGTGTGGG

trpm2cho TTGTGTCCTTCGGAGTGGCCAAGCAGGCC
ATCCTCATCCACAACGAGAGCCGGGTGGACT trpm2hsfr
TGGTGTCCTTCGGGGTGGCCAAGCAGGCCATCCTCATCCACAACGAGCGC
CGGGTGGACT trpm2rat TGGTGTCCTTCGGAGTGGCCAAG
CAGG-CATCCTCATCCACAATGAGAGCCGCGTGGACT

trpm2cho
GGATCTTCCGTGGGGTCGTCTACCACTCTTACCTCACCATCTTTGGGCAG
ATCCCAACCT trpm2hsfr GGCTGTTCCGAGGGGCCGTCTAC
CACTCCTACCTCACCATCTTCGGGCAGATCCCGGGCT trpm2rat
GGATCTTCCGCGGAGTTATCTATCACTCTTACCTTACCATCTTCGG
GCAGATCCCGACCT

trpm2cho ACATTGATGGTGTGAATTTCAGTATGGACCA
GTGCAGCCCTAATGGCACAGACCCCTACA trpm2hsfr
ACATCGACGGTGTGAACTTCAACCCGGAGCACTGCAGCCCCAATGGCACC
GACCCCTACA trpm2rat ACATTGACGGCGTGAATTTCAGC
ATGGACCAGTGCAGCCCCAATGGTACAGACCCCTACA

trpm2cho
AGCCCAAGTGTCCCGAGAGTGACTGGACAGGACAGACACCCGCCTTCCCT
GA-GTGGCTG trpm2hsfr AGCCTAAGTGCCCCGAGAGCGAC
GCGACGCAGCAGAGGCCGGCCTTCCCTGA-GTGGCTG trpm2rat
AGCCCAAGTGTCCTGAGAGTGACTGGACAGGGCAGGCACCCGCCTT
CCCCGAGGTGGCTG

trpm2cho ACGGTCACCTTGCTCTGCCTCTATCT
GCTCTTTGCCAACATCCTGCTGCTCAACCTGCT- trpm2hsfr
ACGGTCCTCCTACTCTGCCTCTACCTGCTCTTCACCAACATCCTGCTGC
TCAACCTCCT- trpm2rat ACAGTCACCCTTCTCTGCCTCT
ACCTGCTCTTCGCCAACATCCTGCTGCTTAATTTGCTC

trpm2cho
CATCGCCATGTTCAACTACACA trpm2hsfr
CATCGCCATGTTCAACTACAC- trpm2rat
CATCGCCATGTTCAACTACACA

54
(No Transcript)
55
Exercice
Trpa1 /ANKTM1

• Can you analyse this protein and design specific
  RT-PCR primers?
• 2. Can you design a molecular tool to answer
  Lauris question?