Development of Primers for Reverse-Transcriptase Polymerase Chain Reaction

Development of Primers for Reverse-Transcriptase Polymerase Chain Reaction

1. Choose your favorite human gene.

2. Find the mRNA or Complementary DNA (cDNA) sequence

-Click on the circle labeled “Gene”

-A new page will come up and again click on the circle labeled “Gene”

-Type in the name of your gene and click Enter

-A number of matches will appear for that gene (Pages 2-3)

-Choose an appropriate selection: it should be human (homo sapiens), it’s an mRNA sequence, and that it’s complete. Each selection has a unique accession number which is sometimes referred to in publications (Pages 4-5)

-There will be a large amount of information that comes up starting with the Summary (Page 6), then sections called “Genomic contect, Genomic regions, Bibliography, Phenotypes, Interactions, General gene info, General protein info, and NCBI reference sequences.”

-Under NCBI Reference sequences you’ll find a section for “mRNA and proteins” (Page 7).

-Click on the mRNA sequence and copy it into a file (Pages 8-9).

3. Determining Primers

-There are many websites that have computer programs that help in choosing primers for PCR including the NCBI website. In addition to the one I listed you can to another website, Pedro’s Molecular Biology Research Tools that lists many websites that are useful in molecular biology,

-We are going to use the website from which we are ordering,

-Click on Scitools. Click on Primer Quest and fill in the necessary information.

-You can cut and paste you sequence in. The numbers are automatically removed.

-Under “Design For:” Pick PCR Primers and Under“Use Parameter Set:” Pick PCR Primers.

-Everything else can be used as a default settings.

-Click “Calculate”

-Choose primer sets that yield a product length between 300-700 bases.

4. Primer Specificity

-The primers should be tested to ensure that they do not anneal other places than where you want.---They can be tested against known DNA sequences, a BLAST search

-This can be done on the right of the page listing your primers or the website is

-Under the database heading click on the “nucleotide collection (nr/nt)” option.

-Enter you sequence and click on the BLAST button

-It should be a short time from seconds to minutes until the results come back

-Check that your favorite gene is at the top of the list in humans and then probably other species, otherwise the primer may amplify an unwanted region of DNA

For the report, please include:

1. A printout with the RNA/DNA sequence and its accession number
2. A printout of the exon map
3. A list of 2 sets of primers and mention at what location they anneal to your sequence or show it directly on the sequence. Also, make sure that you have the annealing temperature of them
4. The first page or two of BLAST results for each of the 4 primers.

Results: 1 to 20 of 90

1.

Sox10

Official Symbol Sox10 and Name: SRY-box containing gene 10 [Mus musculus]

Other Aliases: Dom, Sox21

Other Designations: dominant megacolon; transcription factor SOX-10; transcription factor SOX-M

Chromosome: 15; Location: 15 46.6 cM

Annotation: Chromosome 15, NC_000081.5 (78985343..78994920, complement)

ID: 20665Order cDNA clone

2.

SOX10

Official Symbol SOX10 and Name: SRY (sex determining region Y)-box 10 [Homo sapiens]

Other Aliases: RP5-1039K5.9, DOM, MGC15649, PCWH, WS2E, WS4, WS4C

Other Designations: OTTHUMP00000195094; OTTHUMP00000195097; SRY-related HMG-box gene 10; dominant megacolon, mouse, human homolog of; transcription factor SOX-10

Chromosome: 22; Location: 22q13.1

Annotation: Chromosome 22, NC_000022.10 (38368319..38380539, complement)

MIM: 602229

ID: 6663Order cDNA clone

3.

sox10

Official Symbol sox10 and Name: SRY-box containing gene 10 [Danio rerio]

Other Aliases: cls/sox10, zgc:100757

Other Designations: cls; colorless; colourless; colourless/sox10; golas; gos

Chromosome: 3

Annotation: Chromosome 3, NC_007114.5 (2551636..2560714)

ID: 140616Order cDNA clone

4.

Sox10

Official Symbol Sox10 and Name: SRY (sex determining region Y)-box 10 [Rattus norvegicus]

Other Designations: SRY-box containing gene 10; transcription factor SOX-10

Chromosome: 7; Location: 7q34

Annotation: Chromosome 7, NC_005106.2 (117138694..117149939, complement)

ID: 29361Order cDNA clone

5.

SOX10

SRY (sex determining region Y)-box 10 [Gallus gallus]

Other Aliases: SOX-10

Other Designations: Sry-boxntranscription factor, SOX-10; cSOX10; transcription factor SOX-10

Chromosome: 1

Annotation: Chromosome 1, NC_006088.2 (52999731..53009694)

ID: 395573

SOX10 SRY (sex determining region Y)-box 10 [ Homo sapiens ]

Gene ID: 6663, updated on 29-Mar-2011

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Summary

Official Symbol

SOX10provided by HGNC

Official Full Name

SRY (sex determining region Y)-box 10provided by HGNC

Primary source

HGNC:11190

Locus tag

RP5-1039K5.9

See related

Ensembl:ENSG00000100146;HPRD:03752;MIM:602229

Gene type

protein coding

RefSeq status

REVIEWED

Organism

Homo sapiens

Lineage

Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo

Also known as

DOM; WS4; PCWH; WS2E; WS4C; MGC15649; SOX10

Summary

This gene encodes a member of the SOX (SRY-related HMG-box) family of transcription factors involved in the regulation of embryonic development and in the determination of the cell fate. The encoded protein may act as a transcriptional activator after forming a protein complex with other proteins. This protein acts as a nucleocytoplasmic shuttle protein and is important for neural crest and peripheral nervous system development. Mutations in this gene are associated with Waardenburg-Shah and Waardenburg-Hirschsprung disease. [provided by RefSeq]

NCBI Reference Sequences (RefSeq)

RefSeqs maintained independently of Annotated Genomes

These reference sequences exist independently of genome builds. Explain

These reference sequences are curated independently of the genome annotation cycle, so their versions may not match the RefSeq versions in the current genome build. Identify version mismatches by comparing the version of the RefSeq in this section to the one reported in Genomic regions, transcripts, and products above.

Genomic

1. NG_007948.1RefSeqGene

Range

5001..17221

Download

GenBank, FASTA, Sequence Viewer (Graphics)

mRNA and Protein(s)

1. NM_006941.3 → NP_008872.1transcription factor SOX-10

Status: REVIEWED

Source sequence(s)

BC007595

Consensus CDS

CCDS13964.1

UniProtKB/Swiss-Prot

P56693

Related Ensembl

ENSP00000380093, ENST00000396884

Conserved Domains (2) summary

cd01388
Location:103 – 173
Blast Score: 245

SOX-TCF_HMG-box; SOX-TCF_HMG-box, class I member of the HMG-box superfamily of DNA-binding proteins. These proteins contain a single HMG box, and bind the minor groove of DNA in a highly sequence-specific manner. Members include SRY and its homologs in insects and...

pfam12444
Location:52 – 94
Blast Score: 184

Sox_N; Sox developmental protein N terminal

RefSeqs of Annotated Genomes: Build 37.2

1: AY889970.Reports Synthetic constru...[gi:60656394] / Links

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LOCUS AY889970 1401 bp mRNA linear SYN 29-MAR-2005

DEFINITION Synthetic construct Homo sapiens clone FLH116779.01X SRY-box 10

(SOX10) mRNA, complete cds.

ACCESSION AY889970

VERSION AY889970.1 GI:60656394

KEYWORDS Human ORF Project.

SOURCE synthetic construct

ORGANISM synthetic construct

other sequences; artificial sequences.

REFERENCE 1 (bases 1 to 1401)

AUTHORS Hines,L., Rolfs,A., Jepson,D., Moreira,D., Raphael,J., Kelley,F.,

Shen,B., Halleck,A., Koundinya,M., Hu,Y., Zuo,D., Taycher,E.,

Williamson,J. and LaBaer,J.

TITLE Cloning of human full-length CDS in Creator (TM) recombinational

vector system

JOURNAL Unpublished

REFERENCE 2 (bases 1 to 1401)

AUTHORS Hines,L., Rolfs,A., Jepson,D., Moreira,D., Raphael,J., Kelley,F.,

Shen,B., Halleck,A., Koundinya,M., Hu,Y., Zuo,D., Taycher,E.,

Williamson,J. and LaBaer,J.

TITLE Direct Submission

JOURNAL Submitted (04-JAN-2005) Biological Chemistry and Molecular

Pharmacology, Harvard Institute of Proteomics, 320 Charles St.,

Cambridge, MA02141, USA

COMMENT This CDS clone is a part of a collection of human full-length

expression clones generated by Harvard Institute of Proteomics.

This ORF clone has been cloned with normalized stop-codon. The CDS

has been directionally cloned using BD In-Fusion(TM) cloning system

between the SalI and HindIII sites of the pDNR-Dual vector.

Additional sequences in the clone: 'ACC' after SalI site and

before 'ATG' to provide Kozak consensus sequence. Each clone is

clonally isolated and full-length sequence-verified.

FEATURES Location/Qualifiers

source 1..1401

/organism="synthetic construct"

/mol_type="mRNA"

/db_xref="taxon:32630"

/clone="FLH116779.01X"

/lab_host="Escherichia coli DH5alpha T1 resistant"

/note="derived from MGC template"

gene 1..1401

/gene="SOX10"

CDS 1..1401

/gene="SOX10"

/note="sex determining region Y-box"

/codon_start=1

/transl_table=11

/product="SRY-box 10"

/protein_id="AAX32761.1"

/db_xref="GI:60656395"

/translation="MAEEQDLSEVELSPVGSEEPRCLSPGSAPSLGPDGGGGGSGLRA

SPGPGELGKVKKEQQDGEADDDKFPVCIREAVSQVLSGYDWTLVPMPVRVNGASKSKP

HVKRPMNAFMVWAQAARRKLADQYPHLHNAELSKTLGKLWRLLNESDKRPFIEEAERL

RMQHKKDHPDYKYQPRRRKNGKAAQGEAECPGGEAEQGGTAAIQAHYKSAHLDHRHPG

EGSPMSDGNPEHPSGQSHGPPTPPTTPKTELQSGKADPKRDGRSMGEGGKPHIDFGNV

DIGEISHEVMSNMETFDVAELDQYLPPNGHPGHVSSYSAAGYGLGSALAVASGHSAWI

SKPPGVALPTVSPPGVDAKAQVKTETAGPQGPPHYTDQPSTSQIAYTSLSLPHYGSAF

PSISRPQFDYSDHQPSGPYYGHSGQASGLYSAFSYMGPSQRPLYTAISDPSPSGPQSH

SPTHWEQPVYTTLSRP"

ORIGIN

1 atggcggagg agcaggacct atcggaggtg gagctgagcc ccgtgggctc ggaggagccc

61 cgctgcctgt ccccggggag cgcgccctcg ctagggcccg acggcggcgg cggcggatcg

121 ggcctgcgag ccagcccggg gccaggcgag ctgggcaagg tcaagaagga gcagcaggac

181 ggcgaggcgg acgatgacaa gttccccgtg tgcatccgcg aggccgtcag ccaggtgctc

241 agcggctacg actggacgct ggtgcccatg cccgtgcgcg tcaacggcgc cagcaaaagc

301 aagccgcacg tcaagcggcc catgaacgcc ttcatggtgt gggctcaggc agcgcgcagg

361 aagctcgcgg accagtaccc gcacctgcac aacgctgagc tcagcaagac gctgggcaag

421 ctctggaggc tgctgaacga aagtgacaag cgccccttca tcgaggaggc tgagcggctc

481 cgtatgcagc acaagaaaga ccacccggac tacaagtacc agcccaggcg gcggaagaac

541 gggaaggccg cccagggcga ggcggagtgc cccggtgggg aggccgagca aggtgggacc

601 gccgccatcc aggcccacta caagagcgcc cacttggacc accggcaccc aggagagggc

661 tcccccatgt cagatggtaa ccccgagcac ccctcaggcc agagccatgg cccacccacc

721 cctccaacca ccccgaagac agagctgcag tcgggcaagg cagacccgaa gcgggacggg

781 cgctccatgg gggagggcgg gaagcctcac atcgacttcg gcaacgtgga cattggtgag

841 atcagccacg aggtaatgtc caacatggag acctttgatg tggctgagtt ggaccagtac

901 ctgccgccca atgggcaccc aggccatgtg agcagctact cagcagccgg ctatgggctg

961 ggcagtgccc tggccgtggc cagtggacac tccgcctgga tctccaagcc accaggcgtg

1021 gctctgccca cggtctcacc acctggtgtg gatgccaaag cccaggtgaa gacagagacc

1081 gcggggcccc aggggccccc acactacacc gaccagccat ccacctcaca gatcgcctac

1141 acctccctca gcctgcccca ctatggctca gccttcccct ccatctcccg cccccagttt

1201 gactactctg accatcagcc ctcaggaccc tattatggcc actcgggcca ggcctctggc

1261 ctctactcgg ccttctccta tatggggccc tcgcagcggc ccctctacac ggccatctct

1321 gaccccagcc cctcagggcc ccagtcccac agccccacac actgggagca gccagtatat

1381 acgacactgt cccggcccta a

//

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General Information
Sequence Name : sox10
Batch Date : 4/6/2006 9:27:24 AM
Sequence Length : 1462 /
/ General Options





1 / 100 / 200 / 300 / 400 / 500 / 600 / 700 / 800 / 900 / 1000 / 1100 / 1200 / 1300 / 1462
Primer Set 1
/



1 / 100 / 200 / 300 / 400 / 500 / 600 / 700 / 800 / 900 / 1000 / 1100 / 1200 / 1300 / 1462
/ Set Options


Forward Primer
Primer Sequence: / AGCTCTGGAGGTTGCTGAACGAAA / Options



Primer Start Position: / 472 / Primer Length: / 24
Primer Tm: / 60.4oC / Primer Self Any: / 4.0
Primer GC %: / 50% / Primer Self End: / 0.0
Primer End Stability: / 3.46 / Primer Penalty: / 0.39
Reverse Primer
Primer Sequence: / TGTTGGACATTACCTCGTGGCTGA / Options



Primer Start Position: / 918 / Primer Length: / 24
Primer Tm: / 60oC / Primer Self Any: / 4.0
Primer GC %: / 50% / Primer Self End: / 2.0
Primer End Stability: / 4.26 / Primer Penalty: / 0
Primer Pair/Product
Primer Pair Penalty: / 0.39 / Primer Pair Comp Any: / 5.0
Primer Product Size: / 447bp / Primer Pair Comp End: / 2.0

Primer Set 2
/



1 / 100 / 200 / 300 / 400 / 500 / 600 / 700 / 800 / 900 / 1000 / 1100 / 1200 / 1300 / 1462
/ Set Options


Forward Primer
Primer Sequence: / AAGCTCTGGAGGTTGCTGAACGAA / Options



Primer Start Position: / 471 / Primer Length: / 24
Primer Tm: / 60.4oC / Primer Self Any: / 6.0
Primer GC %: / 50% / Primer Self End: / 1.0
Primer End Stability: / 3.85 / Primer Penalty: / 0.39
Reverse Primer
Primer Sequence: / TGTTGGACATTACCTCGTGGCTGA / Options



Primer Start Position: / 918 / Primer Length: / 24
Primer Tm: / 60oC / Primer Self Any: / 4.0
Primer GC %: / 50% / Primer Self End: / 2.0
Primer End Stability: / 4.26 / Primer Penalty: / 0
Primer Pair/Product
Primer Pair Penalty: / 0.39 / Primer Pair Comp Any: / 5.0
Primer Product Size: / 448bp / Primer Pair Comp End: / 1.0

Taxonomy reports

Distribution of 72 Blast Hits on the Query Sequence

Score E

Sequences producing significant alignments: (bits) Value

gi|33874645|gb|BC018808.2| Homo sapiens SRY (sex determinin... 60 1e-07

gi|55661347|ref|XM_525590.1| PREDICTED: Pan troglodytes sim... 60 1e-07

gi|60653342|gb|AY892449.1| Synthetic construct Homo sapiens... 60 1e-07

gi|60656394|gb|AY889970.1| Synthetic construct Homo sapiens... 60 1e-07

gi|4836840|gb|AF006501.4| Homo sapiens chromosome 22q13.1 C... 60 1e-07

gi|5420148|emb|AL031587.3|HS1039K5 Human DNA sequence from ... 60 1e-07

gi|33988714|gb|BC002824.2| Homo sapiens SRY (sex determinin... 60 1e-07

gi|30179898|ref|NM_006941.3| Homo sapiens SRY (sex determin... 60 1e-07

gi|14043212|gb|BC007595.1| Homo sapiens SRY (sex determinin... 60 1e-07

gi|54696919|gb|BT020029.1| Homo sapiens SRY (sex determinin... 52 3e-05

gi|47678698|emb|CR456584.1| Homo sapiens SOX10 full length ... 52 3e-05

gi|2909359|emb|AJ001183.1|HSJ001183 Homo sapiens mRNA for S... 52 3e-05

gi|49168625|emb|CR536571.1| Homo sapiens full open reading ... 52 3e-05

gi|57093138|ref|XM_538379.1| PREDICTED: Canis familiaris si... 44 0.008

gi|9507130|ref|NM_019193.1| Rattus norvegicus SRY-box conta... 44 0.008

gi|61823560|ref|XM_613423.1| PREDICTED: Bos taurus SRY (sex... 44 0.008

gi|38303848|gb|BC062067.1| Rattus norvegicus SRY-box contai... 44 0.008

gi|2695880|emb|AJ001029.1|RNSOX10 Rattus norvegicus mRNA fo... 44 0.008

gi|19263796|gb|BC025171.1| Mus musculus SRY-box containing ... 42 0.031

gi|17391311|gb|BC018551.1| Mus musculus SRY-box containing ... 42 0.031

gi|51829806|ref|XM_128139.2| PREDICTED: Mus musculus SRY-bo... 42 0.031

gi|3264585|gb|AF047043.1|AF047043 Mus musculus Sox-10 prote... 42 0.031

gi|2826522|gb|AF017182.1|AF017182 Mus musculus putative tra... 42 0.031

gi|1872474|gb|U66141.1|MMU66141 Mus musculus transcription ... 42 0.031

gi|30047226|gb|BC051058.1| Mus musculus SRY-box containing ... 42 0.031

gi|19484027|gb|BC023356.1| Mus musculus SRY-box containing ... 42 0.031

gi|26335594|dbj|AK043220.1| Mus musculus 7 days neonate cer... 42 0.031

gi|26089100|dbj|AK042542.1| Mus musculus 7 days neonate cer... 42 0.031

gi|20196587|emb|AL591921.6| Mouse DNA sequence from clone R... 42 0.031

gi|56550182|emb|AJ585759.1| Musa acuminata partial mRNA for... 36 1.9

gi|39756024|gb|AY400035.1| Homo sapiens HCM0423 gene, VIRTU... 34 7.7

gi|34858949|ref|XM_342542.1| Rattus norvegicus Brain glycog... 34 7.7

gi|46018454|emb|BX935083.2| Gallus gallus finished cDNA, cl... 34 7.7

gi|55649690|ref|XM_524337.1| PREDICTED: Pan troglodytes sim... 34 7.7

gi|55251538|gb|AC104414.8| Mus musculus chromosome 3, clone... 34 7.7

gi|61847177|ref|XM_582759.1| PREDICTED: Bos taurus similar ... 34 7.7

gi|45382254|ref|NM_205418.1| Gallus gallus acetylcholineste... 34 7.7

gi|58531194|dbj|AP008213.1| Oryza sativa (japonica cultivar... 34 7.7

gi|21240682|gb|AC010619.7| Homo sapiens chromosome 19 clone... 34 7.7

gi|57546753|dbj|BA000030.2| Streptomyces avermitilis MA-468... 34 7.7

gi|50754158|ref|XM_414264.1| PREDICTED: Gallus gallus simil... 34 7.7

gi|21664288|emb|AL512357.4|CNS07EF6 Human chromosome 14 DNA... 34 7.7

gi|50284602|gb|AC131593.16| Mus musculus chromosome 3, clon... 34 7.7

gi|34222200|ref|NM_153329.2| Homo sapiens aldehyde dehydrog... 34 7.7

gi|49217583|gb|AC099741.4| Mus musculus strain C57BL6/J chr... 34 7.7

gi|17530773|gb|AC023533.6| Homo sapiens chromosome 8, clone... 34 7.7

gi|16225357|gb|AF417472.1|AF417472 Xenopus laevis cardiac m... 34 7.7

gi|47939922|gb|BC072089.1| Xenopus laevis cDNA clone IMAGE:... 34 7.7

gi|47564195|gb|AC133508.3| Mus musculus BAC clone RP23-383K... 34 7.7

gi|27769115|gb|BC042142.1| Homo sapiens aldehyde dehydrogen... 34 7.7

gi|23272779|gb|BC035641.1| Homo sapiens aldehyde dehydrogen... 34 7.7

gi|34189792|gb|BC014895.2| Homo sapiens aldehyde dehydrogen... 34 7.7

gi|50489695|emb|CR608888.1| full-length cDNA clone CS0DI026... 34 7.7

gi|50483408|emb|CR602601.1| full-length cDNA clone CS0DI087... 34 7.7

gi|13562019|gb|AF350284.1|AF350284 Plectreurys tristis fibr... 34 7.7

gi|3002550|gb|AF022812.1| Dehalospirillum multivorans GTP c... 34 7.7

gi|11908307|gb|AC087255.1|AC087255 Caenorhabditis briggsae ... 34 7.7

gi|34447207|dbj|AP005632.3| Oryza sativa (japonica cultivar... 34 7.7

gi|41151936|gb|AC138548.8| Mus musculus chromosome 3, clone... 34 7.7

gi|331209|gb|M75136.1|IH1CG Ictalurid herpesvirus 1 (channe... 34 7.7

gi|9955987|gb|AY007096.1| Homo sapiens clone TCCCIA00164 mR... 34 7.7

gi|623031|gb|U03472.1|GGU03472 Gallus gallus acetylcholines... 34 7.7

gi|204420|gb|L10668.1|RATGLYPHOA Rat glycogen phosphorylase... 34 7.7

gi|15823967|dbj|AB070940.1| Streptomyces avermitilis oligom... 34 7.7

gi|46878754|emb|AJ698861.1| Gallus gallus partial mRNA for ... 34 7.7

gi|18676619|dbj|AK074136.1| Homo sapiens mRNA for FLJ00209 ... 34 7.7

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Alignments

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gi|33874645|gb|BC018808.2| Homo sapiens SRY (sex determining region Y)-box 10, mRNA (cDNA

clone IMAGE:4861099)

Length = 2897

Score = 60.0 bits (30), Expect = 1e-07

Identities = 30/30 (100%)

Strand = Plus / Plus

Query: 1 agcaggacctatcggaggtggagctgagcc 30

||||||||||||||||||||||||||||||

Sbjct: 276 agcaggacctatcggaggtggagctgagcc 305

gi|55661347|ref|XM_525590.1| PREDICTED: Pan troglodytes similar to SRY (sex determining region

Y)-box 10; dominant megacolon, mouse, human homolog of;

SRY-related HMG-box gene 10 (LOC470206), mRNA

Length = 1122

Score = 60.0 bits (30), Expect = 1e-07

Identities = 30/30 (100%)

Strand = Plus / Plus

Query: 1 agcaggacctatcggaggtggagctgagcc 30

||||||||||||||||||||||||||||||

Sbjct: 11 agcaggacctatcggaggtggagctgagcc 40

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