What have we covered

1
What have we covered?

• Nucleotide and amino acid sequence
  repositories
• NCBI databases tools
• TIGR gene indices TOGA
• InterPro protein domains motifs
• Tools sequence analysis and discovery
• BLAST basic local alignment search tool
• HMMs characterizing a motif
• Multiple Alignment comparing sequences
• GeneFlow managing pedigrees, genotypes
  phenotypes
• NAR database list http//www3.oup.co.uk/nar/datab
  ase/a/

2
KEGG and Primer3
Kyoto Encyclopedia of Genes Genomes Informati
on rich Metabolic pathways http//www.genome.ad.
jp/kegg/ Primer3 (MIT) Primer
design http//www-genome.wi.mit.edu/cgi-bin/prime
r/primer3_www.cgi
3
Why use KEGG?
Developing a gene based marker instead of a
general marker Using a phenotype and knowledge
of the underlying biochemistry to provide a
foundation for your strategy Example Mark
Sorrells, red vs. white kernel color Chalcone
synthase, primary enzyme in anthocyanin
biosynthesis
Using KEGG http//www.genome.ad.jp/kegg/kegg2.htm
l
4
Primer 3

• s Sequence quality is of utmost importance!
• s Avoiding primer dimers self priming
• s Degeneracy of the genetic code makes the job
  difficult
• s Using multiple alignment in conjunction with
  the literature resources to define the regions of
  similarity

http//www-genome.wi.mit.edu/cgi-bin/primer/primer
3_www.cgi
5
Solanum RAMWEIFFVLEQALECIDSTVKQWMATSDSMKD
L-KPQTSSLVSLPDHAFEQPENIMVGR 145 Capsicum
KARRRFRQSLQQVAEDMDHIWKESTKIQDKGKQVSKESLVHDFSSSTNDI
LKVKNNMVGR 155 Saccharum
--------------------------------------------------
---------- Arabidopsis EQRTRMRRRCLSCFGCADYKLC
KKVSAILKSIGELRERSEAIKTDGGSIQVTCREIPIKS 154 Avena
----------------------------------------
-------------------- Aegilops
GGGGGDEASRCRFLKDLDSVASTLSSLLKQAQGSGLPPAVPVPDFDASTL
LQGGHKVFGR 180 Hordeum
-IAHAIKDIQEQLQKVADRRDRNKVFVPHPTRPIAIDPCLRALYAEATEL
VG-----IYG 172
.
  Solanum ENEFEMMLDQLVRG-GRELE---VVSIVGM
GGIGKTTLATKLYSDPY-IMSRFDIRAKAT 200 Capsicum
DDQRKQLLEDLTRSYSGEPK---VIPIVGMGGIGKTTLAKEVYNDES-
ILCRFDVHAWAT 211 Saccharum
------------------------------GGVGKTTFVRSVYNEPQ-VR
GRFRRHAWVT 29 Arabidopsis VVGNTTMMEQVLEFLSEEE
ERG-IIGVYGPGGVGKTTLMQSINNELITKGHQYDVLIWVQ
213 Avena -----------------------------
----GKTTLLHVFNNYLDNKVHDYQVVIFIE 27 Aegilops
NKELNDIVQMLVEPPSPHCTACKVVSIVGFGGLGKTTLAQSVYDDLR-
VKSHFDLRAWAY 239 Hordeum
KRDQDLMRLLSMEGDDASNKRLKKVSIVGFGGLGKTTLARAVYEKIK---
GDFDCRAFVP 229 .
. . .. . .
  Solanum V-----SQEYCVRNVLLGLLSLTSDEPD--
-----DQLADRLQKHLKGRRYLVVIDDIWT 248 Capsicum
I-----SQQHNKKEILLGLLHSTIKMDDRVKMIGEAELADMLQKSLKR
KRYLIVLDDIWS 266 Saccharum
VGA--ASSAAVLKRIMLQVFLERPEMPANADSMEEMQLADTVGRYLSNKP
YLVVLDDLWS 87 Arabidopsis MSR--EFGECTIQQAVGAR
LGLSWDEKETG-----ENRALKIYRALRQKRFLLLLDDVWE
266 Avena VSNSETLNIEEIQQTISERLNLPWNEAEP
I-----AKRAKFLVKALTRKRFVVLLDDVRK 82 Aegilops
VSG--KPDKVELAKQILRSANPRYGGSIDKDAT-FATLQLKLNRLMSS
KRFLIVLDDIWG 296 Hordeum
VGQ-----NPDMKKVLRDILIDLGNPHSDLAMLDANQLIKKLHEFLENKR
YLVIIDDIWD 284 . .
.
  Solanum TKAWDGIKLCFP-----DCYKGSRILLTTR
NVEVAEYA--SSGKPPHHMRLMNFDESWNL 301 Capsicum
CEVWDGVRRCFPT----EDNAGSRILLTTRNDEVACYA--GVENFSLR
MSFMDQDESWSL 320 Saccharum
SDVWDYLSVALP-----DNDLGSRIMVSSRVPDIGSQCRWASAVQVLKHS
PLDHDDSLRL 142 Arabidopsis
EIDLEKTGVPRP-----DRENKCKVMFTTRSIALCNNMG--AEY-KLRVE
FLEKKHAWEL 318 Avena
KFQLEDVGIPTP-----DTNSQSKLILTSRYQDICFQMN--AQRSLIEMQ
ILGNDASWEL 135 Aegilops
DDPFTNEAYNEILSPLRSMESGSRIIAVTQTPKVAGMLD---ASHTYYLN
ALGADDCWSL 353 Hordeum
EKLWEGINFAFSN----RNNLGSRLITTTRIVSVSNSCCSSDGDSVYQME
PLSVDDSRML 340
. . . .  
6
Solanum LHKKIFEKEGS------YSPEFENIGKQIALKC
GGLPLAITVIAGLLS---KISKTLDEW 352Capsicum
FKSAAFSSEA-------LPYEFETVGKQIADECHGLPLTIVVVAGLL---
-KSKRTIEDW 369Saccharum
FRHRAFWSTH--------PPELEEISKEIVRECHGLPLLLVAMGGLMS--
-TKEQAVPAW 191Arabidopsis
FCSKVWRKD-----LLES-SSIRRLAEIIVSKCGGLPLALITLGGAMA--
-HRETEEEWI 369Avena
FLSKLSEEASAAVELLGSQNVIRDYAMAIAQSCGGLPLALNVIGTAVA--
-GLE-ESEWK 191Aegilops
IKESALGGWST---HEESTQELEQIGRKIAAKLNGLPLAAKLMGGLLG--
-ATKSTKYWR 407Hordeum
FSKRIFPDENG------CINEFEQVSRDILKKCGGVPLAIITIASALAGD
QKMKPKCEWD 394
. . . . .... .
 Solanum QNVAENVSSVVSTDLEAKCMRVLALSYHHL
PS-HLKPCFLYFAIFAEDERISVTKLVELW 411Capsicum
KTVAKDVKSFVTNDPDERCSRVLGLSYDHLTS-DLKTCLLHFGIFPED
SDIPVKNLMRSW 428Saccharum
KAVLEELH--RTKDLQLTLPFVLWFAYKDLPS-RLKACFLYFVLFPRTYC
VKRTALIRLW 248Arabidopsis
HASEVLTRFPAEMKGMNYVFALLKFSYDNLESDLLRSCFLYCALFPEEHP
IEIEQLVEYW 429Avena
SAADAIATNMHNIAGVDEMFGRLKYSFDRLTP-TQQQCFLYCTLSPEYGS
ISKDQLVEYW 250Aegilops
IISEKEFSGDITLS-------LLRLSYSYLPG-RLKQCFAFCSIFPKNWK
FDQTNLVRLW 459Hordeum
ILLRSLGSGLTEDNSLEEMRRILSFSYSNLPS-HLKTCLLYLCVYPEDSM
ISRDKLIWKW 453
. .. .
 Solanum AVEGFLNEEEGK-SIEEVAETCINELVDR
SLISIHNLSFDG-KIESCGMHDVTRELCLRE 469Capsicum
MAEGFLKLEN---DLEGEVEKCLQELVDRCLVLVSKRSRDGTKIRSC
KVHDLIYDLCVRE 485Saccharum
IANGYV--------------------------------------------
---------- 254Arabidopsis
VGEGFLTSSNGV-NTIYKGYFLIGDLKAACLLET------GDEKTQVKMY
NVVRSFALWM 482Avena
LAEGFLLNDR------EKGYQIIRSLISACLLQAS-----GSLSSKVKMH
HIIRHLGLWL 299Aegilops
MANGFIQPQSG---TGKRMEDLGTDYFNLLLSRSFFHALRQGRRTHYKMH
DLIHDMAVSA 516Hordeum
VAEGFVHHENQGNSLYLLGLNYFNQLINRSMIQPIYN--YSGEAYACRVH
DMVLDLICNL 511 . ..
.

• The consensus line"" identical or conserved
  residues in all sequences in the alignment""
  indicates conserved substitutions "."
  indicates semi-conserved substitutions.

7
Using Primer3
gtCapsicum GAGCTATTCTGGAGAAATCCTGCAATGTAACGGGCGATCA
TGAGGAGTTAACAATCTTGGAAGTTGAAAT CTTAGAGGTAGCATACACA
ACAGAAGATATGGTTGACTCGGAATCAAGAAGTGTTTTTTTAGCACAGAA
T TTGGAGGAAAGAAACAGGGCTATGTGGGAGATTTTTTTCGTCTTGGAA
CAAGCACTAGAATGCATTGATT CCACCGTGAAACAGTGGATGGCAACAT
CGGACAGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT CAGTTT
ACCTGACCATGCTTTTGAGCAGCCTGAGAATATAATGGTTGGCCGTGAAA
ATGAATTTGAGATG ATGCTGGATCAACTTGTTAGAGGAGGAAGGGAACT
AGAAGTTGTCTCAATCGTAGGGATGGGAGGTATCG GGAAAACAACTTTG
GCTACAAAACTCTATAGTGATCCTTACATTATGTCTCGATTTGATATTCG
TGCAAA AGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCCTA
GGCCTTCTTTCTTTGACAAGTGATGAA CCTGATGATCAACTAGCGGACC
GACTGCAAAAGCATCTGAAAGGCAGGAGATACTTGGTAGTCATTGATG A
CATATGGACTACAAAAGCTTGGGATGGTATAAAACTATGTTTCCCAGACT
GTTATAAGGGAAGCAGAAT ACTCCTGACTACTCGGAATGTGGAAGTGGC
TGAATATGCTAGTTCAGGTAAGCCTCCTCATCACATGCGC CTCATGAAT
TTTGACGAAAGTTGGAATTTACTACACAAAAAGATCTTTGAAAAAGAAGG
TTCTTATTCTC
Regions of Interest (total length840 ) P-loop
364-397 Kinase2 562-598 Exclusion 1,363
398,164 599,240
8
Changing default values

• Max 3 Stability maximum stability of the five
  3 bases.
• high number is desired, no greater than default
  though (9.0)
• max DG for duplex disruption for the five 3'
  bases
• Max Complementarity local alignment
• low number is desired
• SCORE 1.75
• Max 3' Complementarity global alignment
  (3anchored)
• low number is desired
• SCORE 6

5' ATCGNA 3' 3' TA -CGT 5'
5 AGGCTATGGGCCTCGCGA 3'

3' AGCGCTCCGGGTATCGGA 5'
9
Primers