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Primer Design & Restriction Analysis 3rd December 2014

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1 Primer Design & Restriction Analysis 3rd December 2014
Carrie Iwema, PhD, MLS, AHIP Information Specialist in Molecular Biology Health Sciences Library System University of Pittsburgh

2 Goals: PCR primer construction & analysis
Restriction digestion & mapping

3 Tools: Primer Analysis & Design Restriction Mapping NetPrimer
Primer3Plus Primer-BLAST Restriction Mapping NEBcutter Webcutter

4 Primer Analysis & Design
A little something to get you in the mood…

5 Polymerase Chain Reaction (PCR)
1983-Kary Mullis 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

6 Polymerase Chain Reaction (PCR)
Raise temperature (94-98), denature DNA strands Lower temp (50-65), anneal primers Increase temp (72-80), allow time for extensions Repeat process 25-40X

7 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

8 Primer-Dimer formation
SOURCE: NCBI 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

9 Self Dimer (example) The primer sequence is ATCAGCTGTAGAT
SOURCE: NCBI internal dimer 3’ end dimer 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.

10 Cross Dimer (example) Sense primer sequence is ATCAGCTGTAGAT
Anti-sense primer sequence is ATAGTGTAGAT Forms one cross dimer at the 3' end SOURCE: NCBI

11 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

12 Hairpin Loop (example)
SOURCE: NCBI 3’ end hairpin internal hairpin 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

13 Basic Primer Analysis & Design Software
NetPrimer Primer3Plus Primer-BLAST

14 NetPrimer From PREMIER Biosoft Free Major features:
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

15 NetPrimer Enter sequence here

16 NetPrimer—sense primer

17 NetPrimer—help

18 NetPrimer—theories & formulas

19 NetPrimer—antisense primer

20 NetPrimer—antisense hairpin
The most negative (i.e., most stable) DG is used for calculating the rating.

21 NetPrimer—antisense dimer

22 NetPrimer—cross dimer

23 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.

24 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.

25 The higher the rating, the better!
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 = ATTCGCGGATTAGCCGAT DG = cal/mol – ( * -403 cal/°K/mol) = kcal/mol Rating = [(DG dimer * 1.8) + (DG hairpin * 1.4)] Example: [( kcal/mol * 1.8) + (-3.28 * 1.4)] 100 + [ ] 76.76 The higher the rating, the better!

26 NetPrimer—practice primers
Rank these primers with attention to rating, 5’ end DG, and 3’ end stability atgtgcgaggagaaagtgct acaaaccctggacttgcatc cgacttgtcccaggtgtttt ctgaaaccattggcacacac ggctgtgaacatggacattg ggctgaagccaaagctacac

27 NetPrimer Ideal for checking primers
To create primers, try Primer3Plus

28 Primer3Plus Select primer pairs to detect a given template sequence
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

29 Primer3Plus

30 Primer3Plus Design PCR primers to amplify sub region of the sequence (600bp-2600bp) with product size 1800bp-2000bp.

31 Primer3Plus—getting started
click here to retrieve sample sequence, then copy/paste into box

32 Primer3Plus Design PCR primers to amplify sub region of the sequence (600bp-2600bp) with product size 1800bp-2000bp.

33 Primer3Plus Design PCR primers to amplify sub region of the sequence (600bp-2600bp) with product size 1800bp-2000bp.

34 Primer3Plus—results

35 Primer3Plus—results

36 Primer3Plus—results

37 Primer3Plus—Primer3Manager

38 Primer3Plus—check primers

39 Primer3Plus—check primers

40 Primer3Plus—primer info

41 Primer3Plus—BLAST primers

42 Primer3Plus—BLAST primers

43 Primer3Plus—check w/NetPrimer
How good are these primers? Analyze with NetPrimer!

44 Primer3Plus—NetPrimer sense
Left (F) primer

45 Primer3Plus—NetPrimer sense

46 Primer3Plus—NetPrimer antisense
Right (R) primer

47 Primer3Plus—NetPrimer antisense

48 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:

49 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)

50 click here to retrieve sample sequence, then copy/paste into box
Primer-BLAST click here to retrieve sample sequence, then copy/paste into box

51 Primer-BLAST results

52 HSLS MolBio Primer Design Tools

53 Finding Primer Resources…
search.HSLS.MolBio

54 More Primer Databases

55 Restriction Mapping www.biologyreference.com

56 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

57 Restriction Mapping Tools
NEBcutter Webcutter

58 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.

59 NEBcutter

60 NEBcutter—program guide

61 NEBcutter

62 NEBcutter—help

63 NEBcutter—getting started
click here to retrieve sample sequence, then copy/paste into box

64 NEBcutter—restriction map

65 NEBcutter—cutters

66 NEBcutter—zoom in

67 NEBcutter—zoom in more

68 NEBcutter—zoom in more

69 NEBcutter—custom digestion
Get digestion map with SmlI and XbaI

70 NEBcutter—select enzymes

71 NEBcutter—custom digestion map
View gel

72 NEBcutter—agarose gel view

73 NEBcutter—ORF sequence
Find restriction enzymes that will excise the selected portion of the sequence.

74 NEBcutter—ORF sequence

75 NEBcutter—flanking sites

76 NEBcutter—ORF sequence

77 NEBcutter—silent mutagenesis

78 NEBcutter—excise a user-defined sequence

79 NEBcutter—excise a user-defined sequence

80 NEBcutter—enzyme information

81 NEBcutter—enzyme information

82 NEBcutter—REBASE enzyme page

83 REBASE—the restriction enzyme database

84 NEBcutter—enzyme information

85 NEBcutter—methylation sensitivity

86 NEBcutter—generate a vector map

87 NEBcutter—generate a vector map

88 NEBcutter—generate a vector map

89 Sample DNA Sequence You have cloned this mouse sequence:
TGCAGTTTCTATGCAGTTGGTAAAAAGATGCAAAGGAGATGGGAAGGTTGGGAAGGTAAGCCCCACCTCT GAGAACAGAGGCTGGGGTCCAGGCCTGTGGGTGCAAAGGTGCCTCAGCATAGCCAGCATCAGCACACGCA AACCCACTGCCCAAATTTGGGCTCAGGGTTGGCCATTTGCTAGTTCTGCTGCCCTCTTAAGATCTGACTG CCAAATAAATCATCCTCATGTCC ATTGGCGGATCCTGACTACACGCTGTCTTTCTGGCGGAATGGGAAAGTCCAGCACTGCCGCATCCACTCCCGGCAGGATGCT 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 You have cloned this mouse sequence: Answer the questions on the following page using NEBcutter.

90 Sample Exercises What 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.

91 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 cutters If 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

92 Webcutter 2.0 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

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

94 Webcutter Mutate CCGGGT to CCCGGG to introduce Sma I cutting site without changing translation

95 Webcutter—silent mutagenesis
click here to retrieve sample sequence, then copy/paste into box

96 Webcutter—results

97 Webcutter—specific restriction enzymes

98 Thank you! Any questions?
Carrie Iwema Ansuman Chattopadhyay

99 Sequence Manipulation

100 Sequence Manipulation Tools
READSEQ Sequence Manipulation Suite

101 Format your sequence any way you want
READSEQ Format your sequence any way you want

102 READSEQ—change formats
click here to retrieve sample sequence, then copy/paste into box

103 READSEQ—FASTAGenBank

104 Sequence Manipulation Suite

105 removes non-DNA characters from text
SMS—filter DNA removes non-DNA characters from text

106 SMS—reverse complement
converts DNA to its reverse and/or complement counterpart

107 adjusts the spacing of DNA sequences and adds numbering
SMS—group DNA adjusts the spacing of DNA sequences and adds numbering

108 creates a map of the annealing positions of PCR primers
SMS—primer map creates a map of the annealing positions of PCR primers

109 locates regions that match a sequence of interest
SMS—DNA pattern find locates regions that match a sequence of interest

110 finds # of occurrences of each residue
SMS—DNA stats finds # of occurrences of each residue

111 converts DNA sequence into protein
SMS—translate converts DNA sequence into protein


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