Primer Design & Restriction Analysis - PowerPoint Presentation

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Primer Design & Restriction Analysis3rd December 2014

Carrie Iwema, PhD, MLS, AHIPInformation Specialist in Molecular BiologyHealth Sciences Library SystemUniversity of Pittsburghiwema@pitt.eduhttp://www.hsls.pitt.edu/molbio Slide2

Goals:PCR primer construction & analysisRestriction digestion & mapping

http://www.hsls.pitt.edu/molbio Slide3

Tools:Primer Analysis & DesignNetPrimerPrimer3PlusPrimer-BLAST

Restriction MappingNEBcutterWebcutterhttp://www.hsls.pitt.edu/molbio

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Primer Analysis & Design

http://www.hsls.pitt.edu/molbio A little something to get you in the mood…Slide5

Polymerase Chain Reaction (PCR)very simpleexponential amplificationsimilar to natural DNA replication

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

1983-Kary MullisSlide6

Polymerase Chain Reaction (PCR)Raise temperature (94-98), denature DNA strands

Lower temp (50-65), anneal primersIncrease temp (72-80), allow time for extensions

Repeat process 25-40X

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

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Things to consider for primer design…Primer-Dimer formation

Secondary Structures in Primers Illegitimate Priming in Template DNA due to repeated sequencesIncompatibility with PCR conditions

SOURCE: NCBI

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

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Primer-Dimer formationhomology 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 yieldsparticularly problematic when the homology occurs at the 3' end of either primer

SOURCE: NCBI

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

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Self Dimer (example)The primer sequence is ATCAGCTGTAGATIt forms 2 dimers:

internal dimer where 3rd-8th bases of primer in 5‘3' (starting from 5') bond with 6th-11th bases (starting from 3') when primer is placed in reverse direction 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.

3’ end dimer

internal dimer

SOURCE: NCBI

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

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Cross Dimer (example)Sense primer sequence is ATCAGCTGTAGATAnti-sense primer sequence is ATAGTGTAGATForms one

cross dimer at the 3' end

3’ cross dimer

SOURCE: NCBI

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

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Secondary Structure in PrimersHairpin loop formed when primer folds back upon itself held in place by intramolecular bonding

can occur with as few as 3 consecutive homologous bases stability measured by the free energy 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

If free energy <0, the loop could reduce the efficiency of amplification

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

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Hairpin Loop (example)The primer sequence is ATCGATATTCGAAGATIt forms two hairpins:

3' end hairpin where the primer folds back upon itself and first and last 3 bases bond togetherinternal hairpin where 2nd-5th and 9th-12th bases bond together

3’ end hairpin

internal hairpin

SOURCE: NCBI

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

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Basic Primer Analysis & Design SoftwareNetPrimerhttp://www.premierbiosoft.com/netprimer/ Primer3Plus

http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi Primer-BLASThttp://www.ncbi.nlm.nih.gov/tools/primer-blast/

http://www.hsls.pitt.edu/molbio Slide14

NetPrimerhttp://www.premierbiosoft.com/netprimer/From PREMIER

BiosoftFreeMajor features:Primer properties: Tm , molecular weight, GC%, optical activity (both in nmol/A260 & µg/A260), DG, 3' end stability, DH, DS, and 5' end DGSecondary structures: Hairpins, dimers, cross dimers

, palindromes, repeats and runsPrimer rating:

Quantitative prediction of the efficiency of a primerComprehensive 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

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NetPrimer

Enter sequence here

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

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NetPrimer—sense primer

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

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NetPrimer—help

http://www.hsls.pitt.edu/molbio Slide18

NetPrimer—theories & formulas

http://www.hsls.pitt.edu/molbio Slide19

NetPrimer—antisense primer

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

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NetPrimer—antisense hairpin

The most negative (i.e., most stable)

D

G is used for calculating the rating.

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

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NetPrimer—antisense dimer

http://www.hsls.pitt.edu/molbio Slide22

NetPrimer—cross dimer

http://www.hsls.pitt.edu/molbio Slide23

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

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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

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NetPrimer—DG

DG = 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

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NetPrimer—practice primersatgtgcgaggagaaagtgctacaaaccctggacttgcatccgacttgtcccaggtgtttt

ctgaaaccattggcacacacggctgtgaacatggacattgggctgaagccaaagctacachttp://www.hsls.pitt.edu/molbio

Rank these primers with attention to rating, 5’ end

D

G

, and 3’ end stabilitySlide27

NetPrimerIdeal for checking primersTo create primers, try Primer3Plus

http://www.hsls.pitt.edu/molbio Slide28

Primer3Plushttp://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi Select primer pairs to detect a given template sequence

Targets and included/excluded regions can be specifiedSteve 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

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Primer3Plus

http://www.hsls.pitt.edu/molbio Slide30

Primer3Plus

Design PCR primers to amplify sub region of the sequence (600bp-2600bp) with product size 1800bp-2000bp. http://www.hsls.pitt.edu/molbio Slide31

Primer3Plus—getting started

click here to retrieve sample sequence, then copy/paste into box

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

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Primer3Plus

Design PCR primers to amplify sub region of the sequence (600bp-2600bp) with product size 1800bp-2000bp.

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

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Primer3Plus

Design PCR primers to amplify sub region of the sequence (600bp-2600bp) with product size 1800bp-2000bp.

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

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Primer3Plus—results

http://www.hsls.pitt.edu/molbio Slide35

Primer3Plus—results

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

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Primer3Plus—results

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

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Primer3Plus—Primer3Manager

http://www.hsls.pitt.edu/molbio Slide38

Primer3Plus—check primers

http://www.hsls.pitt.edu/molbio Slide39

Primer3Plus—check primers

http://www.hsls.pitt.edu/molbio Slide40

Primer3Plus—primer info

http://www.hsls.pitt.edu/molbio Slide41

Primer3Plus—BLAST primers

http://www.hsls.pitt.edu/molbio Slide42

Primer3Plus—BLAST primers

http://www.hsls.pitt.edu/molbio Slide43

Primer3Plus—check w/NetPrimer

How good are these primers? Analyze with NetPrimer!

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

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Primer3Plus—NetPrimer sense

Left (F) primer

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

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Primer3Plus—NetPrimer sense

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

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Primer3Plus—NetPrimer antisense

Right (R) primer

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

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Primer3Plus—NetPrimer antisense

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

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Primer-BLASThttp://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 Slide49

Primer Design TipsRT-PCR (to avoid unwanted amplification of genomic DNA)Primer pair should span an intronOr

One of the primers should be at exon-exon junctionSNP issuesMay cause mismatch, so pick primers outside of this regionqPCRSpecificity of amplification (amount of PCR product = fluor intensity)

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

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Primer-BLAST

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

here

to retrieve sample sequence, then copy/paste into boxSlide51

Primer-BLAST results

http://www.hsls.pitt.edu/molbio Slide52

HSLS MolBio Primer Design Tools

http://www.hsls.pitt.edu/molbio Slide53

Finding Primer Resources…search.HSLS.MolBio

http://www.hsls.pitt.edu/molbio Slide54

More Primer Databases

http://www.hsls.pitt.edu/molbio Slide55

Restriction Mapping

http://www.hsls.pitt.edu/molbio www.biologyreference.comSlide56

Restriction Mapping—for your sequenceDetermine the # of restriction sitesDetermine the nucleotide position of each cut

List the enzymes that do not cutList the enzymes that cut only once Graphical representation of the restriction sites Textual representation of the restriction sites

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

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Restriction Mapping ToolsNEBcutterhttp://tools.neb.com/NEBcutter2/index.phpWebcutterhttp://bio.biomedicine.gu.se/cutter2/

http://www.hsls.pitt.edu/molbio Slide58

NEBcutter V2.0From New England BioLabsFreeMajor 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

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NEBcutter

http://www.hsls.pitt.edu/molbio Slide60

NEBcutter—program guide

http://www.hsls.pitt.edu/molbio Slide61

NEBcutter

http://www.hsls.pitt.edu/molbio Slide62

NEBcutter—help

http://www.hsls.pitt.edu/molbio Slide63

NEBcutter—getting started

click here to retrieve sample sequence, then copy/paste into box

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

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NEBcutter—restriction map

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

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NEBcutter—cutters

http://www.hsls.pitt.edu/molbio Slide66

NEBcutter—zoom in

http://www.hsls.pitt.edu/molbio Slide67

NEBcutter—zoom in more

http://www.hsls.pitt.edu/molbio Slide68

NEBcutter—zoom in more

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

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NEBcutter—custom digestion

Get digestion map with SmlI and

XbaI

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

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NEBcutter—select enzymes

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

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NEBcutter—custom digestion map

View gelhttp://www.hsls.pitt.edu/molbio

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NEBcutter—agarose gel view

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

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NEBcutter—ORF sequence

Find restriction enzymes that will excise the selected portion of the sequence.

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

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NEBcutter—ORF sequence

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

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NEBcutter—flanking sites

http://www.hsls.pitt.edu/molbio Slide76

NEBcutter—ORF sequence

http://www.hsls.pitt.edu/molbio Slide77

NEBcutter—silent mutagenesis

http://www.hsls.pitt.edu/molbio Slide78

NEBcutter—excise a user-defined sequence

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

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NEBcutter—excise a user-defined sequence

http://www.hsls.pitt.edu/molbio Slide80

NEBcutter—enzyme information

http://www.hsls.pitt.edu/molbio Slide81

NEBcutter—enzyme information

http://www.hsls.pitt.edu/molbio Slide82

NEBcutter—REBASE enzyme page

http://www.hsls.pitt.edu/molbio Slide83

REBASE—the restriction enzyme database

http://www.hsls.pitt.edu/molbio Slide84

NEBcutter—enzyme information

http://www.hsls.pitt.edu/molbio Slide85

NEBcutter—methylation sensitivity

http://www.hsls.pitt.edu/molbio Slide86

NEBcutter—generate a vector map

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

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NEBcutter—generate a vector map

http://www.hsls.pitt.edu/molbio Slide88

NEBcutter—generate a vector map

http://www.hsls.pitt.edu/molbio Slide89

Sample DNA SequenceTGCAGTTTCTATGCAGTTGGTAAAAAGATGCAAAGGAGATGGGAAGGTTGGGAAGGTAAGCCCCACCTCTGAGAACAGAGGCTGGGGTCCAGGCCTGTGGGTGCAAAGGTGCCTCAGCATAGCCAGCATCAGCACACGCAAACCCACTGCCCAAATTTGGGCTCAGGGTTGGCCATTTGCTAGTTCTGCTGCCCTCTTAAGATCTGACTGCCAAATAAATCATCCTCATGTCCATTGGCGGATCCTGACTACACGCTGTCTTTCTGGCGGAATGGGAAAGTCCAGCACTGCCGCATCCACTCCCGGCAGGATGCT

GGGACTCCTAAGTTCTTCTTGACAGATAACCTTGTCTTTGACTCTCTCTATGACCTCATCACACATTATCAGCAAGTACCCCTGCGCTGCAATGAGTTTGAGATGCGCCTTTCAGAGCCTGTTCCACAGACGAATGCCCATGAGAGCAAAGAGTGGTACCACGCAAGCCTGACTAGAGCTCAGGCTGAACATATGCTGATGCGAGTGCCCCGGGATGGGGCCTTCCTGGTGCGGAAACGCAATGAGCCTAACTCATATGCCATCTCTTTCCGGGCTGAGGGAAAGATCAAGCACTGCCGAGTACAGCAGGAAGGCCAGACAGTGATGCTGGGGAACTCTGAGTTTGACAGCCTGGTTGACCTCATCAGCTACTATGAGAAGCACCCCCTGTACCGCAAAATGAAGCTACGCTACCCCATCAACGAGGAGGCACTGGAGAAGATCGGGACAGCTGAACCCGATTATGGGGCACTATACGAGGGCCGCAACCCTGGTTTCTATGTGGAGGCAAACCCTATGCCAACTTTCAAGTGTGCAGTAAAAGCCCTCTTCGACTACAAGGCCCAGAGAGAGGATGAGCTGACCTTCACCAAGAGTGCCATCATCCAGAATGTGGAAAAGCAAGATGGTGGCTGGTGGCGAGGGGACTATGGTGGGAAGAAGCAGCTGTGGTTCCCCTCAAACTATGTGGAAGAGATGATCAATCCAGCAGTCCTAGAGCCTGAGAGGGAGCACCTGGATGAGAACAGCCCACTGGGGGACTTGCTGCGAGGGGTCTTAGATGTGCCAGCTTGTCAGATCGCCATCCGTCCTGAGGGCAAAAACAACCGGCTCTTCGTCTTCTCCATCAGCATGCCATCAGTGGCTCAGTGGTCCCTGGATGTTGCAGCTGACTCACAGGAGGAGTTACAGGACTGGGTGAAAAAGATCCGTGAAGTTGCCCAGACTGCAGATGCCAGGCTCACTGAGGGAAAGATGATGGAGAGGAGGAAGAAGATCGCCTTGGAGCTCTCCGAGCTTGTGGTCTACTGCCGGCCCGTTCCCTTTGATGAAGAGAAGATTGGCACAGAACGTGCTTGTTACCGGGACATGTCCTCCTTTCCGGAAACCAAGGCTGAGA

AGTATGTGAACAAGGCCAAAGGCAAGAAGTTCCTCCAGTACAACCGGCTGCAGCTCTCGCGCATCTACCCTAAGGGCCAGAGGCTAGACTCCTCCAATTATGACCCTCTGCCCATGTGGATCTGCGGTAGCCAGCTTGTA

GCACTCAATTTCCAGACCCCAGACAAGCCTATGCAGATGAACCAGGCCCTCTTCATGGCTGGTGGGCATTGTGGCTATGTGCTGCAGCCAAGCACCATGAGAGACGAAGCCTTTGACCCCTTTGATAAGAGCAGTCTCCGAGGTCTGGAACCCTGTGTCATTTGCATTGAGGTGCTGGGGGCCAGGCATCTGCCGAAGAATGGCCGGGGT

ATTGTGTGTCCTTTTGTGGAGATTGAGGTGGCTGGGGCTGAGTACGACAGCACCAAGCAAAAGACGGAGT

TTGTAGTGGACAACGGACTGAACCCTGTGTGGCCTGCTAAGCCCTTCCACTTCCAGATCAGTAACCCAGA

GTTTGCCTTTCTGCGCTTTGTGGTGTATGAGGAAGACATGTTTAGTGACCAGAACTTCTTGGCTCAGGCT

ACTTTCCCAGTAAAAGGCCTGAAGACAGGATATAGAGCAGTGCCTTTGAAGAACAACTACAGTGAAGACC

TGGAGTTGGCCTCCCTGCTCATCAAGATTGACATTTTCCCTGCTAAGGAGAACGGTGACCTCAGTCCTTT

CAGTGGCATATCCCTAAGGGAACGGGCCTCAGATGCCTCCAGCCAGCTGTTCCATGTCCGGGCCCGGGAA

GGGTCCTTTGAAGCCAGATACCAGCAGCCATTTGAAGATTTCCGCATCTCGCAGGAGCATCTAGCAGACC

ATTTTGACAGTCGGGAACGAAGGGCCCCAAGAAGGACTCGGGTCAATGGAGACAACCGCCTCTAGTCAGA

CCCCACCTAGTTGGAGAGCAGCAGGTGCTGTCCACCTGTGGAATGCCATGAACTGGGTTCTCTGGGAGCT

GTCTACTGTAAAGCCTTCTTGGTCTCACAGCCTGGAGCCTGGATTCCAGCAGTGAAGGCTAGACAAAACC

AAGCCATTAATGATATGTATTGTTTTGGGCCTCCCTGCCCAGCTCTGGGTGAAGGCAAAAAACTGTACTG

TGTCTCGAATTAAGCACACACATCTGGCCCTGAATGTGGAGGTGGGTCCTTCCATCTTGGGCCAGGAGTA

GGGCTGAAGCCCCTTGGAAAGAGAAGTTGCCTCAGTTGGTGGCATAGGAGGTCTCAAGGAGCTGCTGACA

CATTCCTGAAAGAGGAGAAGGAGAAGGAGGAGGAGCCTTGGTGGGCCAGGGAAACAAAGTTTACATTGTC

CTGTAGCTTTAAAACCACAGGGTGAAAGAGTAAATGCCCTGCAGTTTGGCCCTGGAGCCAGGACAGAGGA

ATGCAGGGCCTATAATGAGAAGGCTCTGCTCTGCCCATGGAGGAAGACACAGCACAAGGGCACATTGCCC

ATGGCTGGGTACACTACCCAGCCTGAAAGATACAGGGGATCATGATAAAAATAGCAGTATTAATTTTTTT

TTCTTCTCAGTGGTATTGTAACTAAGTTATTCTGTCCTGCTCCTCACCTTGGAAGGGAAGACCCAGCACA

GAGCCTTTGGGAACAGCAGCTCTATGGGGTGTTGTACTGGGAGAGGGCACTGTCAAGAAGGGTGGAGGGG

CAGGAAGAGAGAAGAGCAATGTCTACCCTGGTGAGCTTTTTTGTTTTTATGACAAAGACGACTCGATATG

CTTCCCCTTAGGAATGGAGATATAGGTAAGTGGAGTCAGGCAGTAGGTACCAAATTAAGCTGCTGCTTGGTGCAGTTTCTATGCAGTTGGTAAAAAGATGCAAAGGAGATGGGAAGGTTGGGAAGGTAAGCCCCACCTCT

GAGAACAGAGGCTGGGGTCCAGGCCTGTGGGTGCAAAGGTGCCTCAGCATAGCCAGCATCAGCACACGCAAACCCACTGCCCAAATTTGGGCTCAGGGTTGGCCATTTGCTAGTTCTGCTGCCCTCTTAAGATCTGACTGCCAAATAAATCATCCTCATGTCC

You have cloned this mouse sequence:

Answer the questions on the following page using

NEBcutter

.

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

Slide90

Sample ExercisesWhat is the %GC content of this Sequence?How many restriction enzymes cut this

sequence only once?If you cut the sequence with Kpn I and Hinc II, how many DNA fragments will be generated?How many open reading frames (ORF) are present?Find the restriction enzymes with compatible ends that can be used to excise the largest ORF.

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

Slide91

Sample Exercises Hints (NEBcutter)What is the %GC content of this Sequence?See top left of page (after entering sequence info)

How many restriction enzymes cut this sequence only once?Select for single cuttersIf you cut the sequence with Kpn I and Hinc II, how many DNA fragments will be generated?

Select Custom digest, then View gel

How many open reading frames (ORF) are present?

Select ORF summary

Find the restriction enzymes

with compatible ends that can

be used to excise the largest ORF.

Select the ORF, then

locate multiple cutters, cut positions

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

Slide92

Webcutter 2.0http://bio.biomedicine.gu.se/cutter2/ FreeMajor features:

Rainbow cutters Highlight your favorite enzymes in color or 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 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

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Webcutter

find alternate versions of the DNA which will translate into the same amino acid sequence, but contains a new restriction site

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

Slide94

Webcutter

Mutate CCGGGT

to CCC

GGG to introduce Sma I cutting site without changing translation

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

Slide95

Webcutter—silent mutagenesis

click here to retrieve sample sequence, then copy/paste into box

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

Slide96

Webcutter—results

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

Slide97

Webcutter—specific restriction enzymes

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

Slide98

Thank you!Any questions?Carrie Iwema

Ansuman Chattopadhyayiwema@pitt.edu ansuman@pitt.edu 412-383-6887 412-648-1297http://www.hsls.pitt.edu/molbio Slide99

Sequence Manipulation

http://www.hsls.pitt.edu/molbio www.vam.ac.uk/images/image/44010-large.jpg Slide100

Sequence Manipulation ToolsREADSEQhttp://www-bimas.cit.nih.gov/molbio/readseq/ Sequence Manipulation Suitehttp://www.bioinformatics.org/sms2/

http://www.hsls.pitt.edu/molbio Slide101

READSEQ

Format your sequence any way you wanthttp://www.hsls.pitt.edu/molbio Slide102

READSEQ—change formats

click here to retrieve sample sequence, then copy/paste into box

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

Slide103

READSEQ—FASTAGenBank

FASTAGenBank

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

Slide104

Sequence Manipulation Suite

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

Slide105

SMS—filter DNA

removes non-DNA characters from text

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

Slide106

SMS—reverse complement

converts DNA to its reverse and/or complement counterparthttp://www.hsls.pitt.edu/molbio Slide107

SMS—group DNA

adjusts the spacing of DNA sequences and adds numbering

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

Slide108

SMS—primer map

creates a map of the annealing positions of PCR primers

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

Slide109

SMS—DNA pattern find

locates regions that match a sequence of interest

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

Slide110

SMS—DNA stats

finds # of occurrences of each residuehttp://www.hsls.pitt.edu/molbio Slide111

SMS—translate

converts DNA sequence into proteinhttp://www.hsls.pitt.edu/molbio