2 nd April 2014
Carrie Iwema, PhD, MLS, AHIP
Information Specialist in Molecular Biology
Health Sciences Library System
University of Pittsburgh iwema@pitt.edu
http://www.hsls.pitt.edu/molbio

Goals:
 PCR primer construction & analysis
 Restriction digestion & mapping http://www.hsls.pitt.edu/molbio

Tools:
 Primer Analysis & Design
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NetPrimer
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Primer3Plus
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Primer-BLAST
 Restriction Mapping
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NEBcutter
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Webcutter http://www.hsls.pitt.edu/molbio

Primer Analysis & Design
A little something to get you in the mood… http://www.hsls.pitt.edu/molbio

Polymerase Chain Reaction (PCR)
1983-Kary Mullis
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 very simple exponential amplification similar to natural DNA replication
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The primary reagents, used in PCR are:
Template DNA – DNA sequence to amplify
DNA nucleotides
– building blocks for new DNA
Taq polymerase – heat stable enzyme catalyzes new DNA
Primers – single-stranded DNA, ~20-50 nucleotides, complimentary to a short region on either side of template DNA http://www.hsls.pitt.edu/molbio

Polymerase Chain Reaction (PCR)
1.
Raise temperature (94-
98), denature DNA strands
2.
Lower temp (50-65), anneal primers
3.
Increase temp (72-80), allow time for extensions
4.
Repeat process 25-40X http://www.hsls.pitt.edu/molbio

Things to consider for primer design…
 Primer-Dimer formation
 Secondary Structures in Primers
 Illegitimate Priming in Template DNA due to repeated sequences
 Incompatibility with PCR conditions
SOURCE: NCBI http://www.hsls.pitt.edu/molbio

Primer-Dimer formation
SOURCE: NCBI
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 homology within a primer ( self dimer ) or between the sense and anti-sense primer ( cross dimer ) bonding of the two primers, increasing primer-dimer artifact and reducing product yields particularly problematic when the homology occurs at the
3' end of either primer http://www.hsls.pitt.edu/molbio

Self Dimer (example) internal dimer
3’ end dimer
SOURCE: NCBI
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The primer sequence is ATCAGCTGTAGAT
It forms 2 dimers:
 internal dimer where 3 rd -8 th bases of primer in 5‘  3' (starting from 5') bond with 6 th -11 th bases (starting from 3') when primer is placed in reverse direction
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3' end dimer where the last 3 bases (starting from 5') of primer placed in 5‘  3' direction bond with last three base (starting from
3') placed in reverse direction.
http://www.hsls.pitt.edu/molbio

Cross Dimer (example)
3’ cross dimer
 Sense primer sequence is ATCAGCTGTAGAT
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Anti-sense primer sequence is ATAGTGTAGAT
Forms one cross dimer at the 3' end
SOURCE: NCBI http://www.hsls.pitt.edu/molbio

Secondary Structure in Primers
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Hairpin loop formed when primer folds back upon itself
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 held in place by intramolecular bonding can occur with as few as 3 consecutive homologous bases
 stability measured by the free energy
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The free energy of the loop is based upon the energy of the intramolecular bond and the energy needed to twist the DNA to form the loop.
If free energy >0 , the loop is too unstable to interfere with the reaction
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If free energy <0 , the loop could reduce the efficiency of amplification http://www.hsls.pitt.edu/molbio

Hairpin Loop (example)
3’ end hairpin
SOURCE: NCBI internal hairpin
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The primer sequence is ATCGATATTCGAAGAT
It forms two hairpins:
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3' end hairpin where the primer folds back upon itself and first and last 3 bases bond together internal hairpin where 2 nd -5 th and 9 th -12 th bases bond together http://www.hsls.pitt.edu/molbio

Basic Primer Analysis & Design Software
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NetPrimer http://www.premierbiosoft.com/netprimer/
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Primer3Plus http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi
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Primer-BLAST http://www.ncbi.nlm.nih.gov/tools/primer-blast/ http://www.hsls.pitt.edu/molbio

NetPrimer http://www.premierbiosoft.com/netprimer/
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 From PREMIER Biosoft
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Free
Major features:
Primer properties : Tm , molecular weight, GC%, optical activity (both in nmol /A260 & µg/A260), DG, 3' end stability,
DH, DS, and 5' end DG
Secondary structures : Hairpins, dimers, cross dimers, palindromes, repeats and runs
Primer rating : Quantitative prediction of the efficiency of a primer
Comprehensive report : Prints complete primer analysis for an individual primer or primer pair
Primer pairs : Analyze individual primers or primer pairs
Comprehensive help : Details all the formulas and references used in primer analysis algorithm http://www.hsls.pitt.edu/molbio

NetPrimer http://www.hsls.pitt.edu/molbio
Enter sequence here

NetPrimer—sense primer http://www.hsls.pitt.edu/molbio

NetPrimer—help http://www.hsls.pitt.edu/molbio

NetPrimer—theories & formulas http://www.hsls.pitt.edu/molbio

NetPrimer—antisense primer http://www.hsls.pitt.edu/molbio

NetPrimer—antisense hairpin http://www.hsls.pitt.edu/molbio
The most negative (i.e., most stable) D G is used for calculating the rating.

NetPrimer—antisense dimer http://www.hsls.pitt.edu/molbio

NetPrimer—cross dimer http://www.hsls.pitt.edu/molbio

NetPrimer—3’ & 5’ stability
An ideal primer has a stable 5' end and an unstable 3' end.
Unstable 3’ = limits bonding to false priming sites. The lower this value, numerically, the more liable the primer is to show secondary bands. less negative = less false priming .
Stable 5’ = called the GC Clamp, it increases bonding to the target site.
The lower this value, numerically, the more efficient is the primer. more negative = better bonding.
http://www.hsls.pitt.edu/molbio

NetPrimer—rating
The rating of a primer provides a quick way of measuring the predicted efficiency of a primer as well as choosing between closely matched primers. The higher the rating of a primer, the higher its amplification efficiency .
http://www.hsls.pitt.edu/molbio

NetPrimer— D G
D G = D H – T * D S = free energy of the primer
D H = enthalpy (internal energy) of primer
T = temperature
D S = entropy (unavailable energy) of primer
Example : primer sequence = ATTCGCGGATTAGCCGAT
D G = -154500 cal/mol – (298.15 * -403 cal/°K/mol) = -34.35 kcal/mol
Rating = 100 + [( D G dimer * 1.8) + ( D G hairpin * 1.4)]
Example : 100 + [(-10.36 kcal/mol * 1.8) + (-3.28 * 1.4)]
100 + [-18.648 + -4.592]
100 + -23.24
76.76
The higher the rating, the better!
http://www.hsls.pitt.edu/molbio

NetPrimer—practice primers
Rank these primers with attention to rating, 5’ end D
G , and 3’ end stability
1.
2.
3.
4.
5.
6.
atgtgcgaggagaaagtgct acaaaccctggacttgcatc cgacttgtcccaggtgtttt ctgaaaccattggcacacac ggctgtgaacatggacattg ggctgaagccaaagctacac http://www.hsls.pitt.edu/molbio

NetPrimer
 Ideal for checking primers
 To create primers, try Primer3Plus http://www.hsls.pitt.edu/molbio

Primer3Plus
 http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi
 Select primer pairs to detect a given template sequence
 Targets and included/excluded regions can be specified
 Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the
WWW for general users and for biologist programmers . In: Krawetz
S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in
Molecular Biology. Human Press, Totowa, NJ, pp 365-386 http://www.hsls.pitt.edu/molbio

Primer3Plus http://www.hsls.pitt.edu/molbio

Primer3Plus
Design PCR primers to amplify sub region of the sequence (600bp-2600bp) with product size 1800bp-2000bp.
http://www.hsls.pitt.edu/molbio

Primer3Plus—getting started click here to retrieve sample sequence, then copy/paste into box http://www.hsls.pitt.edu/molbio

Primer3Plus Design PCR primers to amplify sub region of the sequence
( 600bp-2600bp ) with product size 1800bp-2000bp. http://www.hsls.pitt.edu/molbio

Primer3Plus Design PCR primers to amplify sub region of the sequence
(600bp-2600bp) with product size 1800bp-2000bp . http://www.hsls.pitt.edu/molbio

Primer3Plus—results http://www.hsls.pitt.edu/molbio

Primer3Plus—results http://www.hsls.pitt.edu/molbio

Primer3Plus—results http://www.hsls.pitt.edu/molbio

Primer3Plus—Primer3Manager http://www.hsls.pitt.edu/molbio

Primer3Plus—check primers http://www.hsls.pitt.edu/molbio

Primer3Plus—check primers http://www.hsls.pitt.edu/molbio

Primer3Plus—primer info http://www.hsls.pitt.edu/molbio

Primer3Plus—BLAST primers http://www.hsls.pitt.edu/molbio

Primer3Plus—BLAST primers http://www.hsls.pitt.edu/molbio

Primer3Plus—check w/NetPrimer
How good are these primers?
Analyze with NetPrimer !
http://www.hsls.pitt.edu/molbio

Primer3Plus—NetPrimer sense
Left (F) primer http://www.hsls.pitt.edu/molbio

Primer3Plus—NetPrimer sense http://www.hsls.pitt.edu/molbio

Primer3Plus—NetPrimer antisense
Right (R) primer http://www.hsls.pitt.edu/molbio

Primer3Plus—NetPrimer antisense http://www.hsls.pitt.edu/molbio

Primer-BLAST
 http://www.ncbi.nlm.nih.gov/tools/primer-blast/
 Combines primer design (Primer3) and a specificity check
(BLAST)
 Can also be used w/pre-designed primers
 ref: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3412702/ http://www.hsls.pitt.edu/molbio

Primer Design Tips
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RT-PCR (to avoid unwanted amplification of genomic DNA)
Primer pair should span an intron
Or
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One of the primers should be at exon-exon junction
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SNP issues
May cause mismatch, so pick primers outside of this region
 qPCR
Specificity of amplification (amount of PCR product = fluor intensity) http://www.hsls.pitt.edu/molbio

Primer-BLAST click here to retrieve sample sequence, then copy/paste into box http://www.hsls.pitt.edu/molbio

Primer-BLAST results http://www.hsls.pitt.edu/molbio

HSLS MolBio Primer Design Tools http://www.hsls.pitt.edu/molbio

Finding Primer Resources… search.HSLS.MolBio
http://www.hsls.pitt.edu/molbio

More Primer Databases http://www.hsls.pitt.edu/molbio

Restriction Mapping www.biologyreference.com
http://www.hsls.pitt.edu/molbio

Restriction Mapping—for your sequence
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Determine the # of restriction sites
Determine the nucleotide position of each cut
List the enzymes that do not cut
List the enzymes that cut only once
Graphical representation of the restriction sites
Textual representation of the restriction sites http://www.hsls.pitt.edu/molbio

Restriction Mapping Tools
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NEBcutter http://tools.neb.com/NEBcutter2/index.php
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Webcutter http://bio.biomedicine.gu.se/cutter2/ http://www.hsls.pitt.edu/molbio

NEBcutter V2.0
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From New England BioLabs
Free
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Major features:
Takes a DNA sequence and finds the large, non-overlapping open reading frames using the E. coli genetic code and the sites for all Type II and commercially available Type III restriction enzymes that cut the sequence just once.
By default, only enzymes from NEB are used, but other sets may be chosen.
Further options appear in the output.
Maximum size of input file = 1 MB ; maximum sequence length = 300 KB .
http://www.hsls.pitt.edu/molbio

NEBcutter http://www.hsls.pitt.edu/molbio

NEBcutter—program guide http://www.hsls.pitt.edu/molbio

NEBcutter http://www.hsls.pitt.edu/molbio

NEBcutter—help http://www.hsls.pitt.edu/molbio

NEBcutter—getting started click here to retrieve sample sequence, then copy/paste into box http://www.hsls.pitt.edu/molbio

NEBcutter—restriction map http://www.hsls.pitt.edu/molbio

NEBcutter—cutters http://www.hsls.pitt.edu/molbio

NEBcutter—zoom in http://www.hsls.pitt.edu/molbio

NEBcutter—zoom in more http://www.hsls.pitt.edu/molbio

NEBcutter—zoom in more http://www.hsls.pitt.edu/molbio

NEBcutter—custom digestion
Get digestion map with SmlI and XbaI http://www.hsls.pitt.edu/molbio

NEBcutter—select enzymes http://www.hsls.pitt.edu/molbio

NEBcutter—custom digestion map
View gel http://www.hsls.pitt.edu/molbio

NEBcutter—agarose gel view http://www.hsls.pitt.edu/molbio

NEBcutter—ORF sequence http://www.hsls.pitt.edu/molbio
Find restriction enzymes that will excise the selected portion of the sequence.

NEBcutter—ORF sequence http://www.hsls.pitt.edu/molbio

NEBcutter—flanking sites http://www.hsls.pitt.edu/molbio

NEBcutter—ORF sequence http://www.hsls.pitt.edu/molbio

NEBcutter—silent mutagenesis http://www.hsls.pitt.edu/molbio

NEBcutter—excise a user-defined sequence http://www.hsls.pitt.edu/molbio

NEBcutter—excise a user-defined sequence http://www.hsls.pitt.edu/molbio

NEBcutter—enzyme information http://www.hsls.pitt.edu/molbio

NEBcutter—enzyme information http://www.hsls.pitt.edu/molbio

NEBcutter—REBASE enzyme page http://www.hsls.pitt.edu/molbio

REBASE—the restriction enzyme database http://www.hsls.pitt.edu/molbio

NEBcutter—enzyme information http://www.hsls.pitt.edu/molbio

NEBcutter—methylation sensitivity http://www.hsls.pitt.edu/molbio

NEBcutter—generate a vector map http://www.hsls.pitt.edu/molbio

NEBcutter—generate a vector map http://www.hsls.pitt.edu/molbio

NEBcutter—generate a vector map http://www.hsls.pitt.edu/molbio

Sample DNA Sequence
You have cloned this mouse sequence:
Answer the questions on the following page using NEBcutter.
TGCAGTTTCTATGCAGTTGGTAAAAAGATGCAAAGGAGATGGGAAGGTTGGGAAGGTAAGCCCCACCTCT
GAGAACAGAGGCTGGGGTCCAGGCCTGTGGGTGCAAAGGTGCCTCAGCATAGCCAGCATCAGCACACGCA
AACCCACTGCCCAAATTTGGGCTCAGGGTTGGCCATTTGCTAGTTCTGCTGCCCTCTTAAGATCTGACTG
CCAAATAAATCATCCTCATGTCC
ATTGGCGGATCCTGACTACACGCTGTCTTTCTGGCGGAATGGGAAAGTCCAGCACTGCCGCATCCACTCCC
GGCAGGATGCT
GGGACTCCTAAGTTCTTCTTGACAGATAACCTTGTCTTTGACTCTCTCTATGACCTCATCACACATTATC
AGCAAGTACCCCTGCGCTGCAATGAGTTTGAGATGCGCCTTTCAGAGCCTGTTCCACAGACGAATGCCCA
TGAGAGCAAAGAGTGGTACCACGCAAGCCTGACTAGAGCTCAGGCTGAACATATGCTGATGCGAGTGCCC
CGGGATGGGGCCTTCCTGGTGCGGAAACGCAATGAGCCTAACTCATATGCCATCTCTTTCCGGGCTGAGG
GAAAGATCAAGCACTGCCGAGTACAGCAGGAAGGCCAGACAGTGATGCTGGGGAACTCTGAGTTTGACAG
CCTGGTTGACCTCATCAGCTACTATGAGAAGCACCCCCTGTACCGCAAAATGAAGCTACGCTACCCCATC
AACGAGGAGGCACTGGAGAAGATCGGGACAGCTGAACCCGATTATGGGGCACTATACGAGGGCCGCAACC
CTGGTTTCTATGTGGAGGCAAACCCTATGCCAACTTTCAAGTGTGCAGTAAAAGCCCTCTTCGACTACAA
GGCCCAGAGAGAGGATGAGCTGACCTTCACCAAGAGTGCCATCATCCAGAATGTGGAAAAGCAAGATGGT
GGCTGGTGGCGAGGGGACTATGGTGGGAAGAAGCAGCTGTGGTTCCCCTCAAACTATGTGGAAGAGATGA
TCAATCCAGCAGTCCTAGAGCCTGAGAGGGAGCACCTGGATGAGAACAGCCCACTGGGGGACTTGCTGCG
AGGGGTCTTAGATGTGCCAGCTTGTCAGATCGCCATCCGTCCTGAGGGCAAAAACAACCGGCTCTTCGTC
TTCTCCATCAGCATGCCATCAGTGGCTCAGTGGTCCCTGGATGTTGCAGCTGACTCACAGGAGGAGTTAC
AGGACTGGGTGAAAAAGATCCGTGAAGTTGCCCAGACTGCAGATGCCAGGCTCACTGAGGGAAAGATGAT
GGAGAGGAGGAAGAAGATCGCCTTGGAGCTCTCCGAGCTTGTGGTCTACTGCCGGCCCGTTCCCTTTGAT
GAAGAGAAGATTGGCACAGAACGTGCTTGTTACCGGGACATGTCCTCCTTTCCGGAAACCAAGGCTGAGA
AGTATGTGAACAAGGCCAAAGGCAAGAAGTTCCTCCAGTACAACCGGCTGCAGCTCTCGCGCATCTACCC
TAAGGGCCAGAGGCTAGACTCCTCCAATTATGACCCTCTGCCCATGTGGATCTGCGGTAGCCAGCTTGTA
GCACTCAATTTCCAGACCCCAGACAAGCCTATGCAGATGAACCAGGCCCTCTTCATGGCTGGTGGGCATT
GTGGCTATGTGCTGCAGCCAAGCACCATGAGAGACGAAGCCTTTGACCCCTTTGATAAGAGCAGTCTCCG
AGGTCTGGAACCCTGTGTCATTTGCATTGAGGTGCTGGGGGCCAGGCATCTGCCGAAGAATGGCCGGGGT
ATTGTGTGTCCTTTTGTGGAGATTGAGGTGGCTGGGGCTGAGTACGACAGCACCAAGCAAAAGACGGAGT
TTGTAGTGGACAACGGACTGAACCCTGTGTGGCCTGCTAAGCCCTTCCACTTCCAGATCAGTAACCCAGA
GTTTGCCTTTCTGCGCTTTGTGGTGTATGAGGAAGACATGTTTAGTGACCAGAACTTCTTGGCTCAGGCT
ACTTTCCCAGTAAAAGGCCTGAAGACAGGATATAGAGCAGTGCCTTTGAAGAACAACTACAGTGAAGACC
TGGAGTTGGCCTCCCTGCTCATCAAGATTGACATTTTCCCTGCTAAGGAGAACGGTGACCTCAGTCCTTT
CAGTGGCATATCCCTAAGGGAACGGGCCTCAGATGCCTCCAGCCAGCTGTTCCATGTCCGGGCCCGGGAA
GGGTCCTTTGAAGCCAGATACCAGCAGCCATTTGAAGATTTCCGCATCTCGCAGGAGCATCTAGCAGACC
ATTTTGACAGTCGGGAACGAAGGGCCCCAAGAAGGACTCGGGTCAATGGAGACAACCGCCTCTAGTCAGA
CCCCACCTAGTTGGAGAGCAGCAGGTGCTGTCCACCTGTGGAATGCCATGAACTGGGTTCTCTGGGAGCT
GTCTACTGTAAAGCCTTCTTGGTCTCACAGCCTGGAGCCTGGATTCCAGCAGTGAAGGCTAGACAAAACC
AAGCCATTAATGATATGTATTGTTTTGGGCCTCCCTGCCCAGCTCTGGGTGAAGGCAAAAAACTGTACTG
TGTCTCGAATTAAGCACACACATCTGGCCCTGAATGTGGAGGTGGGTCCTTCCATCTTGGGCCAGGAGTA
GGGCTGAAGCCCCTTGGAAAGAGAAGTTGCCTCAGTTGGTGGCATAGGAGGTCTCAAGGAGCTGCTGACA
CATTCCTGAAAGAGGAGAAGGAGAAGGAGGAGGAGCCTTGGTGGGCCAGGGAAACAAAGTTTACATTGTC
CTGTAGCTTTAAAACCACAGGGTGAAAGAGTAAATGCCCTGCAGTTTGGCCCTGGAGCCAGGACAGAGGA
ATGCAGGGCCTATAATGAGAAGGCTCTGCTCTGCCCATGGAGGAAGACACAGCACAAGGGCACATTGCCC
ATGGCTGGGTACACTACCCAGCCTGAAAGATACAGGGGATCATGATAAAAATAGCAGTATTAATTTTTTT
TTCTTCTCAGTGGTATTGTAACTAAGTTATTCTGTCCTGCTCCTCACCTTGGAAGGGAAGACCCAGCACA
GAGCCTTTGGGAACAGCAGCTCTATGGGGTGTTGTACTGGGAGAGGGCACTGTCAAGAAGGGTGGAGGGG
CAGGAAGAGAGAAGAGCAATGTCTACCCTGGTGAGCTTTTTTGTTTTTATGACAAAGACGACTCGATATG
CTTCCCCTTAGGAATGGAGATATAGGTAAGTGGAGTCAGGCAGTAGGTACCAAATTAAGCTGCTGCTTGG
TGCAGTTTCTATGCAGTTGGTAAAAAGATGCAAAGGAGATGGGAAGGTTGGGAAGGTAAGCCCCACCTCT
GAGAACAGAGGCTGGGGTCCAGGCCTGTGGGTGCAAAGGTGCCTCAGCATAGCCAGCATCAGCACACGCA
AACCCACTGCCCAAATTTGGGCTCAGGGTTGGCCATTTGCTAGTTCTGCTGCCCTCTTAAGATCTGACTG
CCAAATAAATCATCCTCATGTCC http://www.hsls.pitt.edu/molbio

Sample Exercises
1.
What is the %GC content of this Sequence?
2.
How many restriction enzymes cut this sequence only once?
3.
If you cut the sequence with Kpn I and Hinc II, how many DNA fragments will be generated?
4.
How many open reading frames (ORF) are present?
5.
Find the restriction enzymes with compatible ends that can be used to excise the largest ORF.
http://www.hsls.pitt.edu/molbio

Sample Exercises Hints (NEBcutter)
1.
2.
3.
4.
5.
What is the %GC content of this Sequence?
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See top left of page (after entering sequence info)
How many restriction enzymes cut this sequence only once?
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Select for single cutters
If you cut the sequence with Kpn I and Hinc II, how many DNA fragments will be generated?
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Select Custom digest, then View gel
How many open reading frames (ORF) are present?
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Select ORF summary
Find the restriction enzymes with compatible ends that can be used to excise the largest ORF.
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Select the ORF, then locate multiple cutters, cut positions http://www.hsls.pitt.edu/molbio

Webcutter 2.0
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 http://bio.biomedicine.gu.se/cutter2/
Free
Major features:
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Rainbow cutters Highlight your favorite enzymes in color or
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 boldface for easy at-a-glance identification
Silent cutters Find sites which may be introduced by silent mutagenesis of your coding sequence
Sequence uploads Input sequences directly into Webcutter from
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 a file on your hard drive without needing to cut-and-paste
Degenerate sequences Analyze restriction maps of sequences containing ambiguous nucleotides like N, Y, and R.
Circular sequences Choose whether to treat your sequence as linear or circular
Enzyme info Click into the wealth of references and ordering information at New England BioLabs' REBASE, directly from your restriction map results http://www.hsls.pitt.edu/molbio

Webcutter http://www.hsls.pitt.edu/molbio find alternate versions of the
DNA which will translate into the same amino acid sequence, but contains a new restriction site

Webcutter
Mutate CC G GG T to CC C GG G to introduce
Sma I cutting site without changing translation http://www.hsls.pitt.edu/molbio

Webcutter—silent mutagenesis click here to retrieve sample sequence, then copy/paste into box http://www.hsls.pitt.edu/molbio

Webcutter—results http://www.hsls.pitt.edu/molbio

Webcutter—specific restriction enzymes http://www.hsls.pitt.edu/molbio

Any questions?
Carrie Iwema iwema@pitt.edu
412-383-6887
Ansuman Chattopadhyay ansuman@pitt.edu
412-648-1297 http://www.hsls.pitt.edu/molbio

Sequence Manipulation www.vam.ac.uk/images/image/44010-large.jpg http://www.hsls.pitt.edu/molbio

Sequence Manipulation Tools
 READSEQ
 http://www-bimas.cit.nih.gov/molbio/readseq/
 Sequence Manipulation Suite
 http://www.bioinformatics.org/sms2/ http://www.hsls.pitt.edu/molbio

READSEQ
Format your sequence any way you want http://www.hsls.pitt.edu/molbio

READSEQ—change formats click here to retrieve sample sequence, then copy/paste into box http://www.hsls.pitt.edu/molbio

READSEQ—FASTA  GenBank
FASTA http://www.hsls.pitt.edu/molbio
GenBank

Sequence Manipulation Suite http://www.hsls.pitt.edu/molbio

SMS—filter DNA removes non-DNA characters from text http://www.hsls.pitt.edu/molbio

SMS—reverse complement converts DNA to its reverse and/or complement counterpart http://www.hsls.pitt.edu/molbio

SMS—group DNA adjusts the spacing of DNA sequences and adds numbering http://www.hsls.pitt.edu/molbio

SMS—primer map creates a map of the annealing positions of PCR primers http://www.hsls.pitt.edu/molbio

SMS—DNA pattern find locates regions that match a sequence of interest http://www.hsls.pitt.edu/molbio

SMS—DNA stats finds # of occurrences of each residue http://www.hsls.pitt.edu/molbio

SMS—translate converts DNA sequence into protein http://www.hsls.pitt.edu/molbio