Primer Design & Restriction Analysis 2 nd April 2014

Primer Design & Restriction Analysis2nd April 2014

Carrie Iwema, PhD, MLS, AHIPInformation Specialist in Molecular BiologyHealth Sciences Library SystemUniversity of [email protected]

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

mailto:[email protected]


Goals:

PCR primer construction & analysis

Restriction digestion & mapping



Tools:

Primer Analysis & Design NetPrimer Primer3Plus Primer-BLAST

Restriction Mapping NEBcutter Webcutter



Primer Analysis & Design


A little something to get you in the mood…


http://bio-rad.cnpg.com/lsca/videos/ScientistsForBetterPCR/

Polymerase Chain Reaction (PCR) very simple

exponential amplification similar 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


1983-Kary Mullis


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



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



Primer-Dimer formation

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

SOURCE: NCBI



Self Dimer (example)

The primer sequence is ATCAGCTGTAGAT It 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



Cross Dimer (example)

Sense primer sequence is ATCAGCTGTAGAT Anti-sense primer sequence is ATAGTGTAGAT Forms one cross dimer at the 3' end

3’ cross dimer

SOURCE: NCBI



Secondary Structure in Primers Hairpin 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



Hairpin Loop (example)

The primer sequence is ATCGATATTCGAAGAT It forms two hairpins:

3' end hairpin where the primer folds back upon itself and first and last 3 bases bond together

internal hairpin where 2nd-5th and 9th-12th bases bond together

3’ end hairpin

internal hairpin

SOURCE: NCBI



Basic Primer Analysis & Design Software

NetPrimer http://www.premierbiosoft.com/netprimer/

Primer3Plus http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi

Primer-BLAST http://www.ncbi.nlm.nih.gov/tools/primer-blast/


http://www.premierbiosoft.com/netprimer/

http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi

http://www.ncbi.nlm.nih.gov/tools/primer-blast/


NetPrimer http://www.premierbiosoft.com/netprimer/ From PREMIER Biosoft 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.premierbiosoft.com/netprimer/

http://www.premierbiosoft.com/

http://www.premierbiosoft.com/


NetPrimer

Enter sequence here



NetPrimer—sense primer



NetPrimer—help



NetPrimer—theories & formulas



NetPrimer—antisense primer



NetPrimer—antisense hairpin

The most negative (i.e., most stable) DG is used for

calculating the rating.



NetPrimer—antisense dimer



NetPrimer—cross dimer



NetPrimer—3’ & 5’ stabilityAn 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.



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.



NetPrimer—DG DG = DH – T * DS = free energy of the primer

DH = enthalpy (internal energy) of primer T = temperature DS = entropy (unavailable energy) of primer

Example: primer sequence = ATTCGCGGATTAGCCGATDG = -154500 cal/mol – (298.15 * -403 cal/°K/mol) = -34.35 kcal/mol

Rating = 100 + [(DG dimer * 1.8) + (DG hairpin * 1.4)]

Example: 100 + [(-10.36 kcal/mol * 1.8) + (-3.28 * 1.4)]

100 + [-18.648 + -4.592]100 + -23.2476.76

The higher the rating, the better!



NetPrimer—practice primers

1. atgtgcgaggagaaagtgct

2. acaaaccctggacttgcatc

3. cgacttgtcccaggtgtttt

4. ctgaaaccattggcacacac

5. ggctgtgaacatggacattg

6. ggctgaagccaaagctacac


Rank these primers with attention to rating, 5’ end DG, and 3’ end stability


NetPrimer

Ideal for checking primers

To create primers, try Primer3Plus




Primer3Plus


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://jura.wi.mit.edu/rozen/papers/rozen-and-skaletsky-2000-primer3.pdf

http://jura.wi.mit.edu/rozen/papers/rozen-and-skaletsky-2000-primer3.pdf


Primer3Plus



Primer3Plus

Design PCR primers to amplify sub region of the sequence

(600bp-2600bp) with product size 1800bp-2000bp.



Primer3Plus—getting started

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

box


http://files.hsls.pitt.edu/files/molbio/DNA_Analysis_Tools.docx


Primer3PlusDesign PCR primers to amplify sub region of the sequence (600bp-2600bp) with product

size 1800bp-2000bp.



Primer3Plus—results



Primer3Plus—Primer3Manager



Primer3Plus—check primers



Primer3Plus—primer info



Primer3Plus—BLAST primers



Primer3Plus—check w/NetPrimer

How good are these primers? Analyze with

NetPrimer!


http://www.premierbiosoft.com/netprimer/index.html


Primer3Plus—NetPrimer sense

Left (F) primer



Primer3Plus—NetPrimer sense



Primer3Plus—NetPrimer antisense

Right (R) primer



Primer3Plus—NetPrimer antisense



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.ncbi.nlm.nih.gov/pmc/articles/PMC3412702/


Primer Design Tips

RT-PCR (to avoid unwanted amplification of genomic DNA) Primer pair should span an intron

Or One of the primers should be at exon-exon junction

SNP issues May cause mismatch, so pick primers outside of this region

qPCR Specificity of amplification (amount of PCR product = fluor intensity)



Primer-BLAST


click here to retrieve sample sequence,

then copy/paste into box



Primer-BLAST results



HSLS MolBio Primer Design Tools



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

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

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

Finding Primer Resources…

search.HSLS.MolBio




More Primer Databases



http://primerdepot.nci.nih.gov/

http://web.ncifcrf.gov/rtp/gel/primerdb/

http://www.rtprimerdb.org/

http://medgen.ugent.be/methprimerdb/

http://pga.mgh.harvard.edu/primerbank/

http://genepipe.ngc.sinica.edu.tw/primerz/beginDesign.do

http://perlprimer.sourceforge.net/index.html

http://www.bioinformatics.org/primerx/

Restriction Mapping


www.biologyreference.com


Restriction Mapping—for your sequence

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



Restriction Mapping Tools

NEBcutter http://tools.neb.com/NEBcutter2/index.php

Webcutter http://bio.biomedicine.gu.se/cutter2/


http://tools.neb.com/NEBcutter2/index.php

http://bio.biomedicine.gu.se/cutter2/


NEBcutter V2.0 From New England BioLabs Free 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.





NEBcutter



NEBcutter—program guide



NEBcutter



NEBcutter—help



NEBcutter—getting started


box




NEBcutter—restriction map



NEBcutter—cutters



NEBcutter—zoom in



NEBcutter—zoom in more



NEBcutter—custom digestion

Get digestion map with SmlI and XbaI



NEBcutter—select enzymes



NEBcutter—custom digestion map

View gel



NEBcutter—agarose gel view



NEBcutter—ORF sequence

Find restriction enzymes that will excise the

selected portion of the sequence.



NEBcutter—flanking sites



NEBcutter—silent mutagenesis



NEBcutter—excise a user-defined sequence



NEBcutter—enzyme information



NEBcutter—REBASE enzyme page



REBASE—the restriction enzyme database



NEBcutter—enzyme information



NEBcutter—methylation sensitivity



NEBcutter—generate a vector map



Sample DNA SequenceTGCAGTTTCTATGCAGTTGGTAAAAAGATGCAAAGGAGATGGGAAGGTTGGGAAGGTAAGCCCCACCTCT

GAGAACAGAGGCTGGGGTCCAGGCCTGTGGGTGCAAAGGTGCCTCAGCATAGCCAGCATCAGCACACGCAAACCCACTGCCCAAATTTGGGCTCAGGGTTGGCCATTTGCTAGTTCTGCTGCCCTCTTAAGATCTGACTGCCAAATAAATCATCCTCATGTCCATTGGCGGATCCTGACTACACGCTGTCTTTCTGGCGGAATGGGAAAGTCCAGCACTGCCGCATCCACTCCCGGCAGGATGCTGGGACTCCTAAGTTCTTCTTGACAGATAACCTTGTCTTTGACTCTCTCTATGACCTCATCACACATTATCAGCAAGTACCCCTGCGCTGCAATGAGTTTGAGATGCGCCTTTCAGAGCCTGTTCCACAGACGAATGCCCATGAGAGCAAAGAGTGGTACCACGCAAGCCTGACTAGAGCTCAGGCTGAACATATGCTGATGCGAGTGCCCCGGGATGGGGCCTTCCTGGTGCGGAAACGCAATGAGCCTAACTCATATGCCATCTCTTTCCGGGCTGAGGGAAAGATCAAGCACTGCCGAGTACAGCAGGAAGGCCAGACAGTGATGCTGGGGAACTCTGAGTTTGACAGCCTGGTTGACCTCATCAGCTACTATGAGAAGCACCCCCTGTACCGCAAAATGAAGCTACGCTACCCCATCAACGAGGAGGCACTGGAGAAGATCGGGACAGCTGAACCCGATTATGGGGCACTATACGAGGGCCGCAACCCTGGTTTCTATGTGGAGGCAAACCCTATGCCAACTTTCAAGTGTGCAGTAAAAGCCCTCTTCGACTACAAGGCCCAGAGAGAGGATGAGCTGACCTTCACCAAGAGTGCCATCATCCAGAATGTGGAAAAGCAAGATGGTGGCTGGTGGCGAGGGGACTATGGTGGGAAGAAGCAGCTGTGGTTCCCCTCAAACTATGTGGAAGAGATGATCAATCCAGCAGTCCTAGAGCCTGAGAGGGAGCACCTGGATGAGAACAGCCCACTGGGGGACTTGCTGCGAGGGGTCTTAGATGTGCCAGCTTGTCAGATCGCCATCCGTCCTGAGGGCAAAAACAACCGGCTCTTCGTCTTCTCCATCAGCATGCCATCAGTGGCTCAGTGGTCCCTGGATGTTGCAGCTGACTCACAGGAGGAGTTACAGGACTGGGTGAAAAAGATCCGTGAAGTTGCCCAGACTGCAGATGCCAGGCTCACTGAGGGAAAGATGATGGAGAGGAGGAAGAAGATCGCCTTGGAGCTCTCCGAGCTTGTGGTCTACTGCCGGCCCGTTCCCTTTGATGAAGAGAAGATTGGCACAGAACGTGCTTGTTACCGGGACATGTCCTCCTTTCCGGAAACCAAGGCTGAGAAGTATGTGAACAAGGCCAAAGGCAAGAAGTTCCTCCAGTACAACCGGCTGCAGCTCTCGCGCATCTACCCTAAGGGCCAGAGGCTAGACTCCTCCAATTATGACCCTCTGCCCATGTGGATCTGCGGTAGCCAGCTTGTAGCACTCAATTTCCAGACCCCAGACAAGCCTATGCAGATGAACCAGGCCCTCTTCATGGCTGGTGGGCATTGTGGCTATGTGCTGCAGCCAAGCACCATGAGAGACGAAGCCTTTGACCCCTTTGATAAGAGCAGTCTCCGAGGTCTGGAACCCTGTGTCATTTGCATTGAGGTGCTGGGGGCCAGGCATCTGCCGAAGAATGGCCGGGGTATTGTGTGTCCTTTTGTGGAGATTGAGGTGGCTGGGGCTGAGTACGACAGCACCAAGCAAAAGACGGAGTTTGTAGTGGACAACGGACTGAACCCTGTGTGGCCTGCTAAGCCCTTCCACTTCCAGATCAGTAACCCAGAGTTTGCCTTTCTGCGCTTTGTGGTGTATGAGGAAGACATGTTTAGTGACCAGAACTTCTTGGCTCAGGCTACTTTCCCAGTAAAAGGCCTGAAGACAGGATATAGAGCAGTGCCTTTGAAGAACAACTACAGTGAAGACCTGGAGTTGGCCTCCCTGCTCATCAAGATTGACATTTTCCCTGCTAAGGAGAACGGTGACCTCAGTCCTTTCAGTGGCATATCCCTAAGGGAACGGGCCTCAGATGCCTCCAGCCAGCTGTTCCATGTCCGGGCCCGGGAAGGGTCCTTTGAAGCCAGATACCAGCAGCCATTTGAAGATTTCCGCATCTCGCAGGAGCATCTAGCAGACCATTTTGACAGTCGGGAACGAAGGGCCCCAAGAAGGACTCGGGTCAATGGAGACAACCGCCTCTAGTCAGACCCCACCTAGTTGGAGAGCAGCAGGTGCTGTCCACCTGTGGAATGCCATGAACTGGGTTCTCTGGGAGCTGTCTACTGTAAAGCCTTCTTGGTCTCACAGCCTGGAGCCTGGATTCCAGCAGTGAAGGCTAGACAAAACCAAGCCATTAATGATATGTATTGTTTTGGGCCTCCCTGCCCAGCTCTGGGTGAAGGCAAAAAACTGTACTGTGTCTCGAATTAAGCACACACATCTGGCCCTGAATGTGGAGGTGGGTCCTTCCATCTTGGGCCAGGAGTAGGGCTGAAGCCCCTTGGAAAGAGAAGTTGCCTCAGTTGGTGGCATAGGAGGTCTCAAGGAGCTGCTGACACATTCCTGAAAGAGGAGAAGGAGAAGGAGGAGGAGCCTTGGTGGGCCAGGGAAACAAAGTTTACATTGTCCTGTAGCTTTAAAACCACAGGGTGAAAGAGTAAATGCCCTGCAGTTTGGCCCTGGAGCCAGGACAGAGGAATGCAGGGCCTATAATGAGAAGGCTCTGCTCTGCCCATGGAGGAAGACACAGCACAAGGGCACATTGCCCATGGCTGGGTACACTACCCAGCCTGAAAGATACAGGGGATCATGATAAAAATAGCAGTATTAATTTTTTTTTCTTCTCAGTGGTATTGTAACTAAGTTATTCTGTCCTGCTCCTCACCTTGGAAGGGAAGACCCAGCACAGAGCCTTTGGGAACAGCAGCTCTATGGGGTGTTGTACTGGGAGAGGGCACTGTCAAGAAGGGTGGAGGGGCAGGAAGAGAGAAGAGCAATGTCTACCCTGGTGAGCTTTTTTGTTTTTATGACAAAGACGACTCGATATGCTTCCCCTTAGGAATGGAGATATAGGTAAGTGGAGTCAGGCAGTAGGTACCAAATTAAGCTGCTGCTTGGTGCAGTTTCTATGCAGTTGGTAAAAAGATGCAAAGGAGATGGGAAGGTTGGGAAGGTAAGCCCCACCTCTGAGAACAGAGGCTGGGGTCCAGGCCTGTGGGTGCAAAGGTGCCTCAGCATAGCCAGCATCAGCACACGCAAACCCACTGCCCAAATTTGGGCTCAGGGTTGGCCATTTGCTAGTTCTGCTGCCCTCTTAAGATCTGACTGCCAAATAAATCATCCTCATGTCC

You have cloned this mouse sequence:

Answer the questions on the following page

using NEBcutter.



Sample Exercises1. 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.



Sample Exercises Hints (NEBcutter)1. What is the %GC content of this Sequence?

See top left of page (after entering sequence info)2. How many restriction enzymes cut this sequence

only once? Select for single cutters

3. If you cut the sequence with Kpn I and Hinc II, how many DNA fragments will be generated? Select Custom digest, then View gel

4. How many open reading frames (ORF) are present? Select ORF summary

5. 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



Webcutter 2.0 http://bio.biomedicine.gu.se/cutter2/ Free Major 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://bio.biomedicine.gu.se/cutter2/


Webcutter

find alternate versions of the DNA which will

translate into the same amino acid sequence,

but contains a new restriction site



http://rna.lundberg.gu.se/cutter2/

Webcutter

Mutate CCGGGT to CCCGGG to introduce Sma I cutting site without changing

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


Webcutter—silent mutagenesis


box




Webcutter—results



Webcutter—specific restriction enzymes



Thank you!Any questions?

Carrie Iwema Ansuman [email protected] [email protected] 412-383-6887 412-648-1297




http://www.hsls.pitt.edu/guides/genetics

Sequence Manipulation


www.vam.ac.uk/images/image/44010-large.jpg


Sequence Manipulation Tools

READSEQ http://www-bimas.cit.nih.gov/molbio/readseq/

Sequence Manipulation Suite http://www.bioinformatics.org/sms2/


http://www-bimas.cit.nih.gov/molbio/readseq/

http://www.bioinformatics.org/sms2/


READSEQ

Format your sequence any way you want



READSEQ—change formats


box




READSEQ—FASTAGenBank

FASTA GenBank



Sequence Manipulation Suite



SMS—filter DNA removes non-DNA characters from

text



SMS—reverse complementconverts DNA to its reverse

and/or complement counterpart



SMS—group DNA adjusts the spacing of DNA sequences and

adds numbering



SMS—primer mapcreates a map of the annealing

positions of PCR primers



SMS—DNA pattern findlocates regions that match a sequence of

interest



SMS—DNA stats finds # of occurrences of each

residue



SMS—translate converts DNA sequence into

protein



Primer Design & Restriction Analysis 2 nd April 2014

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