BIOINFORMATICS

Primer Design
Primer means an oligonucleotide

BIOLOGI MOLEKULER

Faculty of Biology
Jenderal Soedirman University

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 SOAL
Ada mendapatkan sebuah sekuen nukleotida dari hasil
sekuensing. Anda diminta mencari tahu sekuen tersebut sangat
mirip atau sama dengan sekuen apa (1); Sekuen tersebut
menyandikan asam amino apa saja serta berapa jumlahnya (2);
Bila diminta membuat primer urutan primer seperti apa yang
bisa mendeteksi keberadaan sequen tersebut dan berapa besar
produk yang diharapkan (3); Kemukakan alasan anda (4)
Nucleotide sequence
analyzing
 What is BLAST?
Free, online service from National Center for
Biotechnology Information (NCBI)

http://blast.ncbi.nlm.nih.gov/Blast.cgi
 Introduction

Suppose you has discovered an unknown fragment of DNA

extracted from a gel, which you have had sequenced. You will
want to find out as much as you can about it.

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 Introduction

•Is it contaminated sequences?

•Is it an already known gene?
•Is it related to any other genes either by having a common
ancestor?
•Is it similar in function to other genes via convergent
evolution?
•What could the protein sequence be for this nucleotide
fragment if it is translated and what might this be like?

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 What is BLAST?

Basic Local Alignment Search Tool

 The BLAST algorithm
• The BLAST programs (Basic Local
Alignment Search Tools) are a set of
sequence comparison algorithms
introduced in 1990 that are used to
search sequence databases for optimal
local alignments to a query.
– Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) “Basic local alignment
search tool.” J. Mol. Biol. 215:403-410.
– Altschul SF, Madden TL, Schaeffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ
(1997) “Gapped BLAST and PSI-BLAST: a new generation of protein database
search programs.” NAR 25:3389-3402.

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 BLAST programs
Program Description
Compares an amino acid query sequence against a protein
blastp sequence database.
Compares a nucleotide query sequence against a
blastn nucleotide sequence database.
Compares a nucleotide query sequence translated in all
reading frames against a protein sequence database. You
blastx could use this option to find potential translation products of
an unknown nucleotide sequence.
Compares a protein query sequence against a nucleotide
tblastn sequence database dynamically translated in all reading
frames.
Compares the six-frame translations of a nucleotide query
tblastx
10 sequence against the six-frame translations of a nucleotide
sequence database.
 Running NCBI BLAST

Select Database

Lecture 3.1 11
 Running NCBI BLAST

Select Program

Select Database

Lecture 3.1 12
 Running NCBI BLAST

Copy paste sequence

Select Database

Lecture 3.1 13
 Running NCBI BLAST

Select Database

Click to blasting

Lecture 3.1 14
 BLAST Output

Lecture 3.1 15
 BLAST Output

Lecture 3.1 16
 BLAST Output

Lecture 3.1 17
 BLAST Output

Lecture 3.1 18
 Interpreting Results
• Score: Normalized score of alignment
(substitution matrix and gap penalty). Can be
compared across searches
• Max score: Score of single best aligned
sequence
• Total score: Sum of scores of all aligned
sequences
 Interpreting Results
• Query coverage: What percent of query
sequence is aligned
• E Value: Number of matches with same score
expected by chance. For low values, equal to
p, the probability of a random alignment
• Typically, E < .05 is required to be considered
significant
 BLAST Parameters
• Identities - No. & % exact residue matches
• Positives - No. and % similar & ID matches
• Gaps - No. & % gaps introduced
• Score - Summed HSP score (S)
• Bit Score - a normalized score (S’)
• Expect (E) - Expected # of chance HSP aligns
• P - Probability of getting a score > X
• T - Minimum word or k-tuple score (Threshold)

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 MEANING OF % IDENTITY

• 100% = the sequnce is higly similar

• 97-99% = close similar (indicate new strain)
• 75-90% = novelty (similar function but different specifity)
 TRANSLATE NUCLEOTIDE SEQUENCE

• If your nucleotide sequence can be translated to a peptide

sequence, you probably want to do it (use tool such as ExPASy
Translate Tool)
• Protein blasts are more sensitive and biologically significant

• Sometimes it makes sense to use other blasts

Lecture 3.1 23
TRANSLATE NUCLEOTIDE SEQUENCE

COPY PASTE nucleotide

sequence
TRANSLATE NUCLEOTIDE SEQUENCE

COPY PASTE nucleotide

sequence

Click translate
TRANSLATE NUCLEOTIDE SEQUENCE

COPY PASTE nucleotide

sequence
Right translate

Click translate
 What is primer?

• Oligonucleotides, also referred to as primers, are

short single strands of nucleic acids that are
synthesized from either DNA or RNA in order to bind
to a complementary strand
• Have a target area where they bind and act as the
starting point for polymerase to extend from, and
thus determine what segment of DNA gets amplified

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An important thing when designing primers for PCR
• How many primers are needed,
• The length of the primer (between 15 and 30 base pairs)
• The 5’ and 3’end (the 3’ end of the forward primer will
extend toward the reverse primer)
• The mutation location in primer (put the mutation on the
3’ end of the primer)
• The primer melting/annealing temperature (between
50°C and 65°C)
• The G-C content (base composition of G-C of about 50%-
60%)
• “Primer dimer” ; hairpin; loop; and
• The distance between the forward and reverse primers
(between 300 and 2,000 base pairs apart)
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 1. Open the Gene Ontology from the EBI website at
http://www.ebi.ac.uk/60/ then type “gene of interest"
and then klik "find"

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2. In the window of the website will appear a variety of
menus, then click on" view all” directly "result "

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3. On the screen will display a list of names of species
and their accession, select several accession and open
link in the new tab

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 4. Copy the base sequence boxed "SEQUENCE"
(bottom) along with the names of each accession were
selected, and then paste it into notepad and save the
file with the *.txt format.

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 5. Open the NCBI website (national Centre Of
Biotechnology) at http://www.ncbi.nlm.nih.gov/
address then click on the icon "BLAST"

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6. Then click on the icon "nucleotide blast”

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7. Copy the selected sequence order, then paste in box
"enter the accession number (s), gi (s) or FASTA
sequence (s)"

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 8. In the box "database" select "others", nucleotide
collection (nr/nt) in the "Optimize for" select "somewhat
Similar sequences (BlastN)" then click "BLAST"

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9. Copy the FASTA sequence from the selected
accession into the notepad

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Sequence are selected in notepad, save the file with the *.txt
format

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 10. Open Bioedit program, select the menu "open
sequence set" and select the file you saved earlier, then
click the icon "shade identities and similarities in
alignment windows"

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 Step in primer design by using primer3
http://bioinfo.ut.ee/primer3-0.4.0/
Primer3 (This is the program's main window)

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 Step in primer desaign by using primer3

Step1 – paste the sequence you want to amplify in PCR

into the window.
Step2 – add '<' and '>' and the beginning and end of the
region you want to be amplified (sequence that should
later be sequenced or just for limiting the sequence for
a minimal length of an amplified fragment).
For example:

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If the sequence is

TGGTAGAGACATGCCCCTACCGCCCTGATTGGCTGAAGCTGCGTGCCTGGTGAGGTGACGTGGCTTGCTGTGAGTGGTTGGGGGC
TGAGAGTATATAAGAGTGAGAGATCCGGTGATCGAGGGAGAAAAAGATGAAGCGAGAGAGATGAAGATTGAAGCTTGCTGACTAA
ACTGCTGTTAGAACTGGTGGTCGTGTCGTTCTTGCTGGTTGAGAGCAGACACGACAGTTGCCTACGAGAATGCGATAGTAAGACTG
CATTGCTGAAGACAGCACACATCTTGGTCACAGAATATAAAGAAATAAAACTGAAACTGGCCTGGGAGCGTCCTCCCCATTAGCTAG
CCATCATGACGCATCAATACAACTGACTACTCAGGCATAATATAGTGCAAGAGTGGCCAAGTTTGCCGGGCGTGGTGGCACACGCCT
TTAATCCGAGCACTCGGGAGGCAGAGGAAGGCGAATTTCTGAGTTCAAGGCCAGCCTGGTCTACAAAGTGAGTTCCAGGACAGCC
AGGGCTACACAGAGAAACCCTGTCTCCAAAAACCAAAAAAAAAAAAAAAAATAGTGGCCAAGTTTGTTCCAGAGGTAAGCAACCA
CTTTCTGATTGGATTTGAAGCCAATCCTATGAAGGGATTCCTCTCTGATACTGTAAATCTGGTCAAATCCCATGGTTGGAAAGGCTATA
GGCCCTAGTTGGGAAGCTACGCTGTTCTTTTGGTAAATAGACATCATGTACCCATCAAATTGAAATTAATTAAGTGGGGCTTTTATAG
GCCTATGTCTCTCCTTGCTGCTGGATTAATGCTCCTAAGATAACAGGGGGGAAAAAAACTCTCTGCAGGATTGTTGAACTTCGGAAC
CTCCCTGTCCCAGACAGATTCCTCCTTGGAAGTGGATCTTTTGCTGCAACTCTGTGTCTGATCGATTCCCTTTTAAGGGATAAAAGGA
CTTGGAATCCCTCTAGGGGGAATGCTCTGAACTGCAGTATCAACTGATAACAA

And I'm interested in the sequence in Red. Then '<' and '>' should be added before and after this sequence – for
example –

TGGTAGAGACATGCCCCTACCGCCCTGATTGGCTGAAGCTGCGTGCCTGGTGAGGTGACGTGGCTTGCTGTGAGTGGTTGGGGGC
TGAGAGTATATAAGAGTGAGAGATCCGGTGATCGAGGGAGAAAAAGATGAAGCGAGAGAGATGAAGATTGAAGCTTGCTGACTAA
ACTGCTGTTAGAACTGGTGGTCGTGTCGTTCTTGCTGGTTGAGAGCAGACACGACAGTTGCCTACGAGAATGCGATAGTAAGACTG
CATTGCTGAAGACAGCACACATCTTGGTCACAGAATATAAAGAAATAAAACTGAAACTGGCCTGGGAGCGTCCTCCCCATTAGCTAG
CCATCATGACGCATCAATACAAC<TGACTACTCAGGCATAATATAGTGCAAGAGTGGCCAAGTTTGCCGGGCGTGGTGGCACACGCC
TTTAATCCGAGCACTCGGGAGGCAGAGGAAGGCGAATTTCTGAGTTCAAGGCCAGCCTGGTCTACAAAGTGAGTTCCAGGACAG
CCAGGGCTACACAGAGAAACCCTGTCTCCAAAAACCAAAAAAAAAAAAAAAAATAGTGGCCAAGTTTGTTCCAGAGGTAAGC>A
ACCACTTTCTGATTGGATTTGAAGCCAATCCTATGAAGGGATTCCTCTCTGATACTGTAAATCTGGTCAAATCCCATGGTTGGAAAGG
CTATAGGCCCTAGTTGGGAAGCTACGCTGTTCTTTTGGTAAATAGACATCATGTACCCATCAAATTGAAATTAATTAAGTGGGGCTTTT
ATAGGCCTATGTCTCTCCTTGCTGCTGGATTAATGCTCCTAAGATAACAGGGGGGAAAAAAACTCTCTGCAGGATTGTTGAACTTCG
GAACCTCCCTGTCCCAGACAGATTCCTCCTTGGAAGTGGATCTTTTGCTGCAACTCTGTGTCTGATCGATTCCCTTTTAAGGGATAAA
AGGACTTGGAATCCCTCTAGGGGGAATGCTCTGAACTGCAGTATCAACTGATAACAA
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Step 3 – setting parameters
The program has default parameters that are better to change.
This is the parameter window:

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Step 4 – Picking primers – click on the 'pick primers' button.
The following output will show

• the first primer pair is usually the best one.

• If no primer pairs show, try less stringent conditions in the setting box.
• If the sequence amplified doesn't include your sequence of interest – limit the extra 44
sequence before and after the amplified fragment.
Paste the reverse sequence, then clik “reverse
complement”

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Open website at http://www.operon.com/tools/oligo-
analysis-tool.aspx to check primer dimer

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 Resources for PCR Primer or Oligo Analysis
–IDT OligoAnalyzer 3.0
Name IDT OligoAnalyzer 3.0
Type Web-based software
Key Functions Analyze primer/oligo sequence and structure.
Publication Info Unpublished
Times Cited n/a
Pros Includes analysis of hairpins, self-dimer, heterodimer; customizable
primer/oligo and salt concentrations; many options for various sequence
modifications; direct submission for NCBI BLAST; allows direct order of
primer/oligo;
Cons No evaluation data.
Note
YiBu’s Rating 4 out of 5

Web Site:
http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/

Online Instruction:
http://www.idtdna.com/Analyzer/Applications/Instructions/Default.aspx?AnalyzerInstructions=true
Check Qualty of each Primer
http://sg.idtdna.com/calc/analyzer for checking Hairpin, self-dimer,
hetero-dimer

Copy paste F primer

Copy paste R primer
 Here is Your primer
• Forward
TCGCTAATACCGATGCGGCT

• Reverse
GTGGGAGCATGCTTACTACT

• Reverse compl
AGTAGTAAGCATGCTCCCAC

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Thank you

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