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
Goals:
PCR primer construction & analysis
Restriction digestion & mapping
http://www.hsls.pitt.edu/molbio
Tools: Primer Analysis & Design
NetPrimer Primer3Plus Primer-BLAST
Restriction Mapping NEBcutter Webcutter
http://www.hsls.pitt.edu/molbio
Primer Analysis & Design
http://www.hsls.pitt.edu/molbio
A little something to get you in the mood…
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
http://www.hsls.pitt.edu/molbio
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
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
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
http://www.hsls.pitt.edu/molbio
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
http://www.hsls.pitt.edu/molbio
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
http://www.hsls.pitt.edu/molbio
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
http://www.hsls.pitt.edu/molbio
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
http://www.hsls.pitt.edu/molbio
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.hsls.pitt.edu/molbio
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.hsls.pitt.edu/molbio
NetPrimer—antisense hairpin
The most negative (i.e., most stable) DG is used for
calculating the rating.
http://www.hsls.pitt.edu/molbio
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. 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—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!
http://www.hsls.pitt.edu/molbio
NetPrimer—practice primers
1. atgtgcgaggagaaagtgct2. acaaaccctggacttgcatc3. cgacttgtcccaggtgtttt4. ctgaaaccattggcacacac5. ggctgtgaacatggacattg6. ggctgaagccaaagctacac
http://www.hsls.pitt.edu/molbio
Rank these primers with attention to rating, 5’ end DG, and 3’ end stability
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
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
Primer3PlusDesign PCR primers to amplify sub region of the sequence (600bp-2600bp) with product
size 1800bp-2000bp.
http://www.hsls.pitt.edu/molbio
Primer3PlusDesign PCR primers to amplify sub region of the sequence (600bp-2600bp) with product
size 1800bp-2000bp.
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 antisense
Right (R) primer
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
RT-PCR (to avoid unwanted amplification of genomic DNA) Primer pair should span an intronOr 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)
http://www.hsls.pitt.edu/molbio
Primer-BLAST
http://www.hsls.pitt.edu/molbio
click here to retrieve sample sequence,
then copy/paste into box
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
http://www.hsls.pitt.edu/molbio
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
http://www.hsls.pitt.edu/molbio
Restriction Mapping Tools
NEBcutter http://tools.neb.com/NEBcutter2/index.php
Webcutter http://bio.biomedicine.gu.se/cutter2/
http://www.hsls.pitt.edu/molbio
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.
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—custom digestion
Get digestion map with SmlI and XbaI
http://www.hsls.pitt.edu/molbio
NEBcutter—custom digestion map
View gel
http://www.hsls.pitt.edu/molbio
NEBcutter—ORF sequence
Find restriction enzymes that will excise the
selected portion of the sequence.
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
REBASE—the restriction enzyme database
http://www.hsls.pitt.edu/molbio
Sample DNA SequenceTGCAGTTTCTATGCAGTTGGTAAAAAGATGCAAAGGAGATGGGAAGGTTGGGAAGGTAAGCCCCACCTCT
GAGAACAGAGGCTGGGGTCCAGGCCTGTGGGTGCAAAGGTGCCTCAGCATAGCCAGCATCAGCACACGCAAACCCACTGCCCAAATTTGGGCTCAGGGTTGGCCATTTGCTAGTTCTGCTGCCCTCTTAAGATCTGACTGCCAAATAAATCATCCTCATGTCCATTGGCGGATCCTGACTACACGCTGTCTTTCTGGCGGAATGGGAAAGTCCAGCACTGCCGCATCCACTCCCGGCAGGATGCTGGGACTCCTAAGTTCTTCTTGACAGATAACCTTGTCTTTGACTCTCTCTATGACCTCATCACACATTATCAGCAAGTACCCCTGCGCTGCAATGAGTTTGAGATGCGCCTTTCAGAGCCTGTTCCACAGACGAATGCCCATGAGAGCAAAGAGTGGTACCACGCAAGCCTGACTAGAGCTCAGGCTGAACATATGCTGATGCGAGTGCCCCGGGATGGGGCCTTCCTGGTGCGGAAACGCAATGAGCCTAACTCATATGCCATCTCTTTCCGGGCTGAGGGAAAGATCAAGCACTGCCGAGTACAGCAGGAAGGCCAGACAGTGATGCTGGGGAACTCTGAGTTTGACAGCCTGGTTGACCTCATCAGCTACTATGAGAAGCACCCCCTGTACCGCAAAATGAAGCTACGCTACCCCATCAACGAGGAGGCACTGGAGAAGATCGGGACAGCTGAACCCGATTATGGGGCACTATACGAGGGCCGCAACCCTGGTTTCTATGTGGAGGCAAACCCTATGCCAACTTTCAAGTGTGCAGTAAAAGCCCTCTTCGACTACAAGGCCCAGAGAGAGGATGAGCTGACCTTCACCAAGAGTGCCATCATCCAGAATGTGGAAAAGCAAGATGGTGGCTGGTGGCGAGGGGACTATGGTGGGAAGAAGCAGCTGTGGTTCCCCTCAAACTATGTGGAAGAGATGATCAATCCAGCAGTCCTAGAGCCTGAGAGGGAGCACCTGGATGAGAACAGCCCACTGGGGGACTTGCTGCGAGGGGTCTTAGATGTGCCAGCTTGTCAGATCGCCATCCGTCCTGAGGGCAAAAACAACCGGCTCTTCGTCTTCTCCATCAGCATGCCATCAGTGGCTCAGTGGTCCCTGGATGTTGCAGCTGACTCACAGGAGGAGTTACAGGACTGGGTGAAAAAGATCCGTGAAGTTGCCCAGACTGCAGATGCCAGGCTCACTGAGGGAAAGATGATGGAGAGGAGGAAGAAGATCGCCTTGGAGCTCTCCGAGCTTGTGGTCTACTGCCGGCCCGTTCCCTTTGATGAAGAGAAGATTGGCACAGAACGTGCTTGTTACCGGGACATGTCCTCCTTTCCGGAAACCAAGGCTGAGAAGTATGTGAACAAGGCCAAAGGCAAGAAGTTCCTCCAGTACAACCGGCTGCAGCTCTCGCGCATCTACCCTAAGGGCCAGAGGCTAGACTCCTCCAATTATGACCCTCTGCCCATGTGGATCTGCGGTAGCCAGCTTGTAGCACTCAATTTCCAGACCCCAGACAAGCCTATGCAGATGAACCAGGCCCTCTTCATGGCTGGTGGGCATTGTGGCTATGTGCTGCAGCCAAGCACCATGAGAGACGAAGCCTTTGACCCCTTTGATAAGAGCAGTCTCCGAGGTCTGGAACCCTGTGTCATTTGCATTGAGGTGCTGGGGGCCAGGCATCTGCCGAAGAATGGCCGGGGTATTGTGTGTCCTTTTGTGGAGATTGAGGTGGCTGGGGCTGAGTACGACAGCACCAAGCAAAAGACGGAGTTTGTAGTGGACAACGGACTGAACCCTGTGTGGCCTGCTAAGCCCTTCCACTTCCAGATCAGTAACCCAGAGTTTGCCTTTCTGCGCTTTGTGGTGTATGAGGAAGACATGTTTAGTGACCAGAACTTCTTGGCTCAGGCTACTTTCCCAGTAAAAGGCCTGAAGACAGGATATAGAGCAGTGCCTTTGAAGAACAACTACAGTGAAGACCTGGAGTTGGCCTCCCTGCTCATCAAGATTGACATTTTCCCTGCTAAGGAGAACGGTGACCTCAGTCCTTTCAGTGGCATATCCCTAAGGGAACGGGCCTCAGATGCCTCCAGCCAGCTGTTCCATGTCCGGGCCCGGGAAGGGTCCTTTGAAGCCAGATACCAGCAGCCATTTGAAGATTTCCGCATCTCGCAGGAGCATCTAGCAGACCATTTTGACAGTCGGGAACGAAGGGCCCCAAGAAGGACTCGGGTCAATGGAGACAACCGCCTCTAGTCAGACCCCACCTAGTTGGAGAGCAGCAGGTGCTGTCCACCTGTGGAATGCCATGAACTGGGTTCTCTGGGAGCTGTCTACTGTAAAGCCTTCTTGGTCTCACAGCCTGGAGCCTGGATTCCAGCAGTGAAGGCTAGACAAAACCAAGCCATTAATGATATGTATTGTTTTGGGCCTCCCTGCCCAGCTCTGGGTGAAGGCAAAAAACTGTACTGTGTCTCGAATTAAGCACACACATCTGGCCCTGAATGTGGAGGTGGGTCCTTCCATCTTGGGCCAGGAGTAGGGCTGAAGCCCCTTGGAAAGAGAAGTTGCCTCAGTTGGTGGCATAGGAGGTCTCAAGGAGCTGCTGACACATTCCTGAAAGAGGAGAAGGAGAAGGAGGAGGAGCCTTGGTGGGCCAGGGAAACAAAGTTTACATTGTCCTGTAGCTTTAAAACCACAGGGTGAAAGAGTAAATGCCCTGCAGTTTGGCCCTGGAGCCAGGACAGAGGAATGCAGGGCCTATAATGAGAAGGCTCTGCTCTGCCCATGGAGGAAGACACAGCACAAGGGCACATTGCCCATGGCTGGGTACACTACCCAGCCTGAAAGATACAGGGGATCATGATAAAAATAGCAGTATTAATTTTTTTTTCTTCTCAGTGGTATTGTAACTAAGTTATTCTGTCCTGCTCCTCACCTTGGAAGGGAAGACCCAGCACAGAGCCTTTGGGAACAGCAGCTCTATGGGGTGTTGTACTGGGAGAGGGCACTGTCAAGAAGGGTGGAGGGGCAGGAAGAGAGAAGAGCAATGTCTACCCTGGTGAGCTTTTTTGTTTTTATGACAAAGACGACTCGATATGCTTCCCCTTAGGAATGGAGATATAGGTAAGTGGAGTCAGGCAGTAGGTACCAAATTAAGCTGCTGCTTGGTGCAGTTTCTATGCAGTTGGTAAAAAGATGCAAAGGAGATGGGAAGGTTGGGAAGGTAAGCCCCACCTCTGAGAACAGAGGCTGGGGTCCAGGCCTGTGGGTGCAAAGGTGCCTCAGCATAGCCAGCATCAGCACACGCAAACCCACTGCCCAAATTTGGGCTCAGGGTTGGCCATTTGCTAGTTCTGCTGCCCTCTTAAGATCTGACTGCCAAATAAATCATCCTCATGTCC
You have cloned this mouse sequence:Answer the
questions on the following page
using NEBcutter.
http://www.hsls.pitt.edu/molbio
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.
http://www.hsls.pitt.edu/molbio
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
http://www.hsls.pitt.edu/molbio
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://www.hsls.pitt.edu/molbio
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
Webcutter
Mutate CCGGGT to CCCGGG to introduce Sma I cutting site without changing
translationhttp://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—specific restriction enzymes
http://www.hsls.pitt.edu/molbio
Thank you!Any questions?
Carrie Iwema Ansuman [email protected] [email protected] 412-383-6887 412-648-1297
http://www.hsls.pitt.edu/molbio
Sequence Manipulation
http://www.hsls.pitt.edu/molbio
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.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
SMS—filter DNA removes non-DNA characters from
text
http://www.hsls.pitt.edu/molbio
SMS—reverse complementconverts 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 mapcreates a map of the annealing
positions of PCR primers
http://www.hsls.pitt.edu/molbio
SMS—DNA pattern findlocates 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