UNI
Design of PCR Primers
fGdCTTCTG CTCAATCTTT ctACAACCAA AGCTCTGTCT TGAA> GTCAI UU I I U I ÚUACUAI U ATCATGTTTT CCTTGATATC ATGT "GCTTCAACA CTCCAAATAC AGAGGTAATT AAATATTATT ATCA <TATAATATG TTATTGATTT TTTGTTTGTG ATTTCATTTA GATTT "CTATGATTT CTTAGCATGA AATACAATTT TTGGAGAAAC AACT TTAAAAACA AAACTTGAAT TTTGAGAAAT TCAAAGATGT TATA' "GTCAAAATT TAACAATTAT TCTTCTAAAT CATCCGGATT CCGT 3TACACATCT ACAATTTTCA ATTGAGGTAT TCTTGTTTTG ATGC 3ACGAATAGT TTGATTGATA AAAAAAATTC TAACCAATAT GATA 3TTTATTTTC TTTTTGTCAA ACCATACTTT ATACTATGTA ACTTT 3AGATTATTG AAAATAGTTT ATTTATAAAA TAGTAACCTA TTGT1 VAAAAAAAAA AAAAAATTGT AAATCGTGTT TGCAAACGAC ATG1 'CTTAGTTTp AAACTAGCTG ATAtTctTcA AATCGACTGT TCTT>
kATCAACC/Vi TTAQOATOAA TQAQAAmAA TTGTAAACAC TTCA 0"GGTGATTT TAAAGAATAT GTTTTACTTA TGTTATGAAC TATC" TGTGAAATA TTTCATAACT AATGTGGAAA ACTATATAAC CCCT ^AAACGTAAG TAAAATTTAT GAAATCCTAT CA1 1 I I I AAA GGTT/ <TCAAAAAGT AATAATTCTT GGTACTTGCA ATA I I I I I GT CATT/ "AGTTTATTA ATTTTATTTT GATTAAATGG TTTTAGATCC ATCAG 3AGATCGCAG TTATAGCTGT AGACGATCCG AAGAAAGCAT TA1 ^AAAATTCAA CGAGACAATA TAGATCTCAT AATCACAGAT TAT1 CTGGTATGAA CGGTTTACAA CTCAAAAAAC AAATCACTCA GGA rfVATTTACCGG TCTTAGGTAA CATTTTTTGT TCTTTACAAC TTAA
Hana Konečná
CEITEC Central European Institute of Technology NCBR National Centre for Biomolecular Research
UNI
definition
aplications
modifications
synthesis
purification
quality control
design of sequence rules
software OLIGO 7 example
MUNI
oligonucleotide
• short single stranded structure
• DNA or RNA (also PNA)
• hydroxyl on both ends
(no phosphate on 5-end as usual)
oligonucleotide
orientation! polymerase!
5'end
3' end
CH H
MUNI
Aplications of synthetic oligonucleotides
• primers for synthesis of complementary DNA
PCR, Real-Time PCR
• gene synthesis and recombinant proteins
• hybridisation probes for cloning
• site directed mutagenesis
• sequencing ang genetic profiling
• diagnostics - tests and biosensors
• gene arrays
• blocation of gene expression - antisense oligo
• prospective therapeutics and DNA vaccines
• NMR monitoring of DNA - protein interactions
• structural X-ray analysis of NA
MUNI
Modifications
• degeneration
• end of sequence
• bases
• phosphate
• carbohydrate
• PNA
Additional resources for interested https://www. glenresearch. com/
I
Degenerated oligonucleotides
ACG TAC GTA CGT ACG TAC non-degenerated
ACG TAM GTA CGT ACG TAC M = A/C
ACG TAC GTA CDT ACG TAC D = A/G/T
ACG TAC GTA CGT ACG NAC N = A/C/G/T
I
Degenerated oligonucleotides
2-deoxyinosin
M	AorC
R	AorG
W	AorT
S	CorG
Y	CorT
K	G orT
V	A or C or G
H	A or C orT
D	A or G orT
B	C orG orT
N	G or A or Tor C
X	G or A or Tor C
UNI
postsynthetic modifications
sequencing fragmenation analysis gene arrays Real-Time PCR
	Phosphorylation
-►	Amino group
-►	Thio group
	Digoxigenin
-►	Biotin
	Enzymes
	Psoralen
	Acridine
	Cholesterole
-►	Fluorescent dyes
	Quenchers
	2,4- dinitrophenyl
	Spacer
	Branching
	Blocation
* Additional resources for interested https://www. glenresearch. com/
MUNI
Modifications on 3'- end
Phosphate
derivatized matrix Thio group
—►  Amino group Spacer Acridine —► Biotin —►   Fluorescent dyes —► Quenchers Cholesterole 2,4 dinitrophenyl
* Additional resources for interested https://www.Qlenresearch.com/
MUNI
• 2x labeled probe
• REPORTER
• QUENCHER
rawwflRD ppimem
2, Strand displacement: When the probe is intact, the reporter dye emission is quenched.
r. a1
5_9,
r I'
3, Cleavage: During each extension cycle, the DNA polymerase cleaves the reporter dye from the probe.
SV J"
—^
B'
9'
4, Polymerization completed: Once separated from the quencher, the reporter dye emits its characteristic fluorescence.
I'
Additional resources for interested https://www. glenreseorch.com/
MUNI
Other modifications
Phosphorothioates Phosphorodithioates H-phosphonates Methylphosphonates
backbone
carbohydrate
Modifications in 2'- position Ribose modification
* Additional resources for interested https://www. glenresearch.com/
MUNI
Therapeutics
+ Nondegradable by nucleases! Modification of phosphodiester
I
0 = P
I
o
0 =
0
1
p
I
o
CH
phosphorothioate
methylphosphonate phosphoramidate
CH2 H3C-N 0 = P-BHa"
3 -thioformacetal      methylene(methyliminio) boranophosphate
* Additional resources for interested https://www. glenresearch. com/
UNI
antisense oligonucleotide
oligonucleotide or analogue complementary to segment of rna or dna inhibition of normal function due to coupling
Nucleus \ mRNA
Translation
Ribosome Protein
Cytoplasm
Cell membrane
3te
Antigene (triplex) Ribozyme
Antisense
Sense/Aptamer
RNA with catalytic activity
MUNI
Peptidonucleic acid
noncharged molecule binding with DNA/RNA
N-(2-aminoethyl)-glycine
•NH O
OH
H,N-
PNA
DNA
PNA
DNA
UNI
Why PNA
thermostable
Tm not depending on salts high specificity high affinity
resistant towards enzymes
* Additional resources for interested Gambari R. Expert Opin Ther Pat. 2014, 24(3):267-94. Peptide nucleic acids: a review on recent patents and technology transfer.
UNI
LNA
Locked Nucleic Acid
• 2'-0, 4'-C methylene bridge
• supressed flexibility of ribofuranose ring
• structure is locked into rigid C3-endo conformation
• enhanced hybridisation
• outstanding biostability
* Additional resources for interested httos://www. alenresearch. com/ https://www.ncbi. nlm,nih.gov/pmc/articles/PMC3412486/
LNA-based Oligonucleotide Electrotransfer for miRNA Inhibition. Molecular Therapy (2012); 20 8, 1590-1598
MUNI
design synthesis
purification
EXPEDITE 8909
UNI
Oligonucleotide Synthesis
• no enzyme
• organic synthesis on solid matrix
• adds new base in cycles
• from 3'-end to 5'-end
• in anhydrous environment
ii
ch^ c-0->-0-«
T
Step 3. Capping
dmt- O
- O- p _o -, - o
OR N Stop 4. Oxidation
O
ii
cmj- c - o    - e
Step 3. Capping
Liquid Chromatography
UNI
LC option: Perfusion Chromatography
classical sorbent POROS
1341ong.bio - 20.0ul
0.15H
2 6 0 n m
0.10-
0.05^
0.00-
0510    15    20    25    30 35
Min <--W
Min
134.bio - 20.0ul
0.1W
2 6 0 ■ m
O.IOJ
0.05H
forot &C(	
	
*        i  i —e/-	
0.0
2.5 Min
M U l\l I    Other tools for Quality Control
LC- M S * additional literature for interested
An automated compliance-ready LC-MS workflow for mass confirmation of both modified and unmodified oligonucleotides, Sep 2020 https://www.waters.com/webassets/cms/library/docs/720006820en.pdf
PAGE
Mast/cbarge
Capillary electrophoresis trace
MUNI
YIELD
Yield
85      85     75      75      65 65 crude purified crude purified crude purified
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PURIFICATION
• Sephadex
• RP cartridge
• HPLC
190.bio - 20-Ovl
1.1 U III
• manual
• computer assisted
* Additional literature for interested
https://www.sciencedirectxom/science/article/pii/S2001037019300844 www.protocol-online.org/prot/Research Tools/Online Tools/Oligo Design/index.html
Main features of good PCR primer sequence
• highly specific
• no dimers and hairpins
• stable duplexes with active sequence
• lightly unstable 3'-end
UNI
OLIGO 6
PCR primers hybridisation probes sequencing primers
OLIGO 7 (from 2008)
TaqMan probes primers for nested PCR molecular beacons siRNA
* Additional resources for interested http://oligo.net/tutorials.html
MUNI
Terminology of PCR primers	
forward primer... part of the + string reverse primer... part of the - string	
	pos:     350      tm: 57.1
	113           ,3€0 .370
CCTGGCTCTGACTACTCAjr h   h   h   h......h......h......►   ►   ►   ► i	1   1   1  1  1  1   1   1   1   1   1   1   1   1   1  1  1   1   1   1   1   1   1   1   1   1   1 1 hhhhhhthhth
	
TTAATG C CTG G CTGTGACTACTCACWtt EAAGGATGGTATTGAG C CTATGTG G G AAG A GAGAAAAACAAAC GGGGAGGAC GATGG CTAATTACATTGAACAAACAH ACACACG AAGTGAtCTC	
AATTAC G GAC C GACACTGATGAGTGAAAAATTC CTAC CATAACTC G GATACAC C CTTCTACTC11111GTTTG C C C CTC CTG CTAC C GATTAATGTAACTTGTTTGTC GTCTCTG CTTCACTG GAG	
	
LMPGtDYSLFKLJ-GIEPMiELJEKNK   1   T~ Uli         1    \ 1	NKQQRR5DL
UPPER - FORWARD - LEFT
5'-►
+ 5' 3' -  3'-5'
<-5'
LOWER - REVERSE - RIGHT
MUNI
Terminology
forward primer., reverse primer..
part of the + string part of the - string
pos:
350
tm:
57.1
, 2E€ _          , 270 _	, 2G0 _        ,,290,,          ^33             1.3             120             110 !'-■)	110             100 170
		
		
TTAATG C CTG G CTGTGACTACTCAC1	111IAAGGATGGTATTGAGCCTATCTGGGAAGATGACAAAAACAAACGGGGmGGACGA GGCTAATTACATTGAACAAACA<KACACACGAAGTGACC C	
AATTAC GGACC G AC ACTG ATG AGTG AAAAATTC CTAC C ATAACTC G G ATAC AC C CTTCTACTCTTTTTGTTTG CCCCTCCTGCTACC GATTAATGTAACTTGTTTGTC GTCTCTG CTTCACTG GAG		
4                   4                   4                   1                   4 H	* ACTC G GATACAC CCTTCTACTC	
		
LMPGCDY5L	FKDCIEPHWEDEKNKRGGRW        I   T L	NKQQRR5DL
UPPER - FORWARD - LEFT
5'-► polymerase
+ 5] ^^^^=1 3'
polymerase * 5'
LOWER - REVERSE - RIGHT
MUNI
Annealing of PCR primers
pos:
350
tm: 57.1
	, 2G0 _          , 29D _           300             Ö.3             3?0             33Ü 3^0	110 170
		
		
TTAATGC CTG G CTGTGACTACTCAC t	111IAAGGATGGTATTGAGCCTATCTGGGAAGATGACAAAAACAAACGGGGAGGACGA GGCTAATTACATTGAACAAAtAGCAGAGACGAAGTGACC C	
AATTAC GGACCGACACTGATGAGTGAAAAATTCCTAC CATAACTCGGATACACCCTTCTACTCTTTTTGTTTG CCCCTCCTGCTACCGATTAATGTAACTTGTTTGTCGTCTCTG CTTCACTG GAG		
4                   4                   4                   1                   4 H	< ACTC G GATACAC CCTTCTACTC	
		
LMPGCDY5 L	F   K   D   G   I   E   P   M   W   E   DE   KNKRGGRW       I   T L	NKQQRR5DL
UPPER - FORWARD - LEFT
+ 5'-- 3'-
3' 5'
5' LOWER - REVERSE - RIGHT
MUNI
5' CTT CTG CTC AAT CTT TCT AC 3' FORWARD
read from left to right
+ C ' 1 ATGdCTTCTG CTCAATCTTT CTACf\ACCAA AGCTCTGTCT TGAAAATCAA
51 TGTCAI (JMJ I I (J I I (J AI ÜATGTTTT CCTTGATATC ATGTCACGCA
101 TGCTTCAACA CTCCAAATAC AGAGGTAATT AAATATTATT ATCATATTAT
151 ATATAATATG TTATTGATTT TTTGTTTGTG ATTTCATTTA GATTTTTATT
201 TCTATGATTT CTTAGCATGA AATACAATTT TTGGAGAAAC AACTAGCAGT
251 TTTAAAAACA AAACTTGAAT TTTGAGAAAT TCAAAGATGT TATATATATA
301 TGTCAAAATT TAACAATTAT TCTTCTAAAT CATCCGGATT CCGTTTACAT
351 GTACACATCT ACAATTTTCA ATTGAGGTAT TCTTGTTTTG ATGCCTTTGA
401 GACGAATAGT TTGATTGATA AAAAAAATTC T A AC CA AT AT GATATATAAA
451 GTTTATTTTC TTTTTGTCAA ACCATACTTT ATACTATGTA ACTTTTTTAA
501 GAGATTATTG AAAATAGTTT ATTTATAAAA TAGTAACCTA TTGTTGAATT
551 AAAAAAAAAA A a a a a ATraT a a ArarraTT rar, a a a r.n Ar. ATGTGATTTA
601 TCTTAGTTTA /|A ACT AG CTG ATATTCTTCAlAATCGACTGT TCTTATAAGT
651 AATCAACCAA T IAUUAIUAA I UAUAAI AAA TTGTAAACAC TTCAATGAAA
701 ATGGTGATTT TAAAGAATAT GTTTTACTTA TGTTATGAAC TATCTCAAAT
751 TTGTGAAATA TTTCATAACT AATGTGGAAA ACTATATAAC CCCTCCATAC
801 AAAACGTAAG TAAAATTTAT GAAATCCTAT CATTTTTAAA GGTTAAACCA
851 ATCAAAAAGT AATAATTCTT GGTACTTGCA ATATTTTTGT CATTATATTT
901 TAGTTTATTA ATTTTATTTT GATTAAATGG TTTTAGATCC ATCAGTTATG
951 GAGATCGCAG TTATAGCTGT AGACGATCCG AAGAAAGCAT TATCTACTCT 1001 AAAAATTCAA CGAGACAATA TAGATCTCAT AATCACAGAT TATTATATGC 1051 CTGGTATGAA CGGTTTACAA CTCAAAAAAC AAATCACTCA GGAATTTGGA 1101 AATTTACCGG TCTTAGGTAA CATTTTTTGT TCTTTACAAC TTAAATTAAA
3'
5'TGAAGAATATCAGCTAGTTT 3' REVERSE
read from right to left complementary
1.1 U III
«oe
Sequence
DNA Sequence				selected Oligo	Position	Lcngtn^Ü
Sequence Length:	LS6S nt		m	Forward Primer	259	IS
Reading Frame:	+ 1	ID		ifia£rse Primer	32S ^^Jä^r	
Current Oligo Length:	21 nt		B	UpperUngo^™		
Position:	356	ID	_B_	Lower Oligo	294	22_
ID W	593°C	ID		PGR Product	S7nt	
#	Feature	Location
1	source	-IE..1S5Ö-
	CD5	1..651
		
=1
m
pos: I    350      tm: | 57,1
.........
,270 ,230 I i.........i I I I
,290 ,300 i.........i......
,310
,320
,330 ,340 I I I i.........i.......
350
,3E0 ,370 i.........i......
CC~CGC~C~CAC~AC~C^
TTAATG C CTG G CTCTC AtTAtTt AtTTTTTAAG GATG GTATT G AG C CTATGTG G G AAG ATGAG AAAAACAAAC G G G GAG GAC GA~G G C^PTTAC ATTG AAC
AATTAC GGACC G AC ACTG ATG AGTG AAAAATTC CTAC CATAACTC GGATACACC CTTCTACTCTTTTTGTTTG C C C CTC CTG CT^yfrTAATGTAACTTGTTTGTC GTCTCTG CTTCACTG GAG
.........ACTC G GATACACCCTTCTACTC ^
....................... CCTCCTGCTACC GATTAA
I
LMPGCDYSLFKDGIEPMWEDEKHKRGGRW ITL
NKQQRR5DL
_
I
Search for Primers & Probes
7
—
Search Options Subsearches
Search in: iVf + Strand 5? - Strand Search Mode: @ Select Verify
5? Complex Substrate
^ PCR Primers. Compatible with the _ Forward Primer   O Reverse Primer
0 TaqMan Probes & PCR Pairs Compatible with the      Upper Probe
w Lower Probe
O Molecular Beacons & PCR Pairs O Nested Primers
Sequencing Primers C Hybridization Probes
siRNA Probes
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I
Search for Primers & Probes
Search Options ~ Subsearches 1
Search method: Compatible Pairs
0 Eliminate Ambiguous Bases
0 Duplex-free Oligonucleotides
0 Highly Specific Oligonucleotides (3'-end Stability)
_ 5*-end Stability
_ siRNA Internal Stability
0 Oligonucleotides with CC Clamp
Q Oligonucleotides within Selected Tm Limits Hairpin-free Oligonucleotides
5! Eliminate Mono- and Di-Nucleotide Repeats
0 Detect Sequence Repeats
B Eliminate Frequent Oligonucleotides
Omit High Secondary Structure Regions in the Template Check Primers/Probe Sequence Constraints
0 Restrict the Number of C Bases
0 Eliminate False Priming Oligonucleotides
and_     Continue Above Search in Other File(s)
_ Consensus Primers
I	Search	)
		
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( Parameters ) ( Ranges ) ( Defaults )
MUNI
File:
oo
PGR
File: Human 4E.seq
Optimal Annealing Temperature: 50.S T (Max: 66.3 °Q
Position and Length
Uppe Lower Oligo
	CC	P.E#	Score
7B.9	29.6	n/a	697
56.9	45.5	47L/47L	S40
55.3	29.6	4S9 / 4S9	S34
55.5	33.3	479 / 479	9L7
55.4	39.1	45? f 451	S4L
Product Tm - Reverse Primer Tm: Z3.6 Primers Tm difference: L.6 °C
Comments:
	Concentration	
Forward Primer	200.0	nM
Reverse Primer	20 0.0	nM
Upper Oligo	200.0	nM
Lower Oligo	200.0	nM
Monovalent Cation	50.0	mM
Free Mg[2+|	0.7	mM
Total Na[+] Equivalent: mM
MUNI
©60
Selected Primers
File: BRCA2 gene.seq
AY436640:1543SF22	
5' CA ATA TATA C C CTA CTC C C CTA 3'	
Length:	22-mer
5core:	802 points
5' Position:	L5438
WW 53.4	52.6 °C
AC/Ag (25 °C)\ -30.5	-29.2 kcal/rnol
A5/As: -472.1	-449.5 cal/°K*mol
AH/Ah: -L7L.3	-L63.2 kcal/rnol
3'AC:	-6.5 kcal/niol
Degeneracy: ^^^^^	
P.E.#:	^43/4431
1/E:	^.Iffnni ol /A2 G0
31.1 pg/A260	
AY436640:15917R20	
5' CACCTACATATTACCCCACA 3'	
Length:	20-mer
Score:	914 points
3' Position:	15917
Tm/tm: 53.1	53.S °C
AC/Ag (25 ÜC): -28.6	-28.5 kcal/rnol
AS/As: -430.5	-419.6 cal/°K * mol
AH/Ah: -157.0	-153.6 kcal/rnol
3'AC:	-6.9 kcal/rnol
Degeneracy:	
P.E.#:	^77/47^
1/E:	^^*9!ff?nni ol /A^ q n
31.0 ug/A2C0	
Priming Efficiency pe Score
UNI
Secondary structures
eoe	Current Oligo Duplexes	
file: BRCA2 gene.acq		
Current Oligo 2L-mer [5042]
[Currentt Oligo! - The most stable 3-dinier: # of hydrogen bonds = 10: AC = -0t7 kcal/mol
5 cAArTACArAAA^cAM^ra a- DIMER intermolecular
3' ArTAAACrTAAATAC-ATTAAC:-
[Current- Oligo] - The most stable 3-dinier: * of hydrogen bonds = L0: AC = -7.3 kcal/mol: Tm = 2.9°C
5 1  TAArPrGAATTTATCTAATTC 3 f
I I I I I I I I I I
3'   G TTAATG TATTTAAU TTTAAT 5"
The most stable dimer overall: & of hydrogen bonds = L0: AC = -7.4 kcal/mol: Tm = 2.2irC
S1   UAATTAGArAAATTCAAATTA 3'
I I I I I I I I I I
3 '  ATTAAAC TTAAATAt]ATTAAt] 5 1
Hairpin: loop = 5 nt: AC = -3.0 kcal/mol; Tm = 54.6 °C
5'?ttrTfiGA HAIRPIN intramolecular
3 ' ATTAAACTTAAAT-1
MUNI
ooo
Current Oligo Hairpin Stems
File: BRCA2 gene.seq
Current Oligo 2L-nner [5042J
L # of paired bases = 5; loop = 5 nt; AG = -3.0 kcal/mol; Tm = 54.6 *C		
5042 GAA1 1 1 1 5056 CTTJ	5046 \A 5052	5 r GAATTÄG-, III A 3 'ATTAAACTTAAAT-1
		
2. # of paired bases = 6; loop = 5 nt; AC = 0.2 kcal/mol; Tm = 2L7 *C		
5043 AATTi 1 1 5061 TTAAJ	\ZA 5049   5 1GAATTAG ATA-, II               II Ma LCT 5055 1 3 r ATTAAACTTA-1	
		
3. # of paired bases = 4: loop = 2 nt; AC = 0.9 kcal/mol; Tm = 8.7 T		
5052 AAT 1 1 5061 TTA	r 5055 A 5058	5 1 GAATTAGATAAATTC-. 1 1 1 1 3 r A!?T AAA-
III
668   Reverse Primer False Priming Sites I File: M13MP18 \
Reverse Primer M13MP18:6310R19 (positive strand) * Priming efficiency of the perfect match is 482 (above the threshold) ▼
Priming efficiency 482 (above the threshold)
5'(6328)   GGTTTTCCCAGTCACGACG (6310)3'
i i i i i i i i i i i i i i i i i i i 3'(6328) ccaaaagggtcagtgctgc (6310)5*
Priming efficiency 244 (above the threshold)
5'(6328)   GGTTTTCCCAGTCACGACG (6310)3'
iii   i i i i mum 3'(626)  agcaaatggtc—tgctgc (610)5'
Priming efficiency 193 (above the threshold)
5'(6328)   GGTTTTCCCAGTCACGACG (6310)3'
iii     i i i i   iii iii 3'(5125)  Ictaagtggtcagtg-tgc (5108)5'
© ^ O     Forward Primer Composition
File: BRCA2 gene.seq
Forward Primer AY436640 6275F19
	64.2°	(nearest neighbor method]
	56.5#	(nearest neighbor method]
Tm	70.8°	[%CC method)
Tm	56#	(2(A+T)° + 4(C + C)0 method)
Tm(RNA)(lM Na]	81*	|*CC method)
Tm (DNA. RNA)[ 1M Na)	74.7°	[*GC method)
A260/A280	1.59	(single strand]
Molecular Weight	S.8K	(one strand)
Molecular Weight	11.7K	(two strands]
ug/OD	47.4 IdsDNA]	
Base	Number**	
A	2	(10.5*1
C	5	(26.3*]
C |	4	(21.1%)
T	8	(42.1*)
A + T	10	(S2.6*|
G + C	9	(47.4*1
MUNI
file: NewDatabase.cdb
O'iqor'Lcleotide Database
5?
Si
□
# of Records: 29
	#	Date	ID Number	Sequence	3'-Dim.	AC	P.E / p.e.		Tm /tm			,1
	1											
□	21	12/02/06	AY436640:5916R19	AA 1CCC 1 CCC 1 1 1 AL IL It,	_	SC	430	430	54.1	54.5		
□	22	12/02/06	AY436640:5916R2O	CAATGCCTGCCTCTAGTCTG ^	0.3	sc	366	450	SO.9	57.2		T
□	23	12/02/06	AY436640:5937R21	TCAA 1 1 1 L 1 1 1AGCTTCCCAT \—		laň	J49	449	54.7	53. L		
	24	12/02/06	AY436640:5937R22	TTCAA 1 1 1 L 1 1 1AGCTTCCCAT		■HEI	Bss	45S	SS.9	53.S		
u	25	12/02/06	AY436640:4695U22	TCCCTTAA CAAAA CTAATCCAT	0.3	sc	432	432	54.5	53.0		
u	26	12/02/06	AY436640:5325U22	AATTACCTC111CTTATCCCAA	0.3	sc	453	453	53.3	53.0		0
u	27	12/02/06	AY436640:5786L23	CTCTCCCTA CAA CATTATCA CTC	-0.3	sc	4SI	45L	54.S	55.0		A
u	2S	12/02/06	AY436640:5S60LL9	AACAACCAAACCCAACCTC	-0.9	sc	444	444	55.3	55.9		T
Oligonucleotide 5et& (64)
□
□
□ U U
□
36 42 47 39 33 61 4S
Forward Primer
S S
9 9 9 9
Rever&e Primer
—T~
23
24 L4
15
16 L7 LS
Upper Ol i go 3
25 25 25 25 25 25 2S
Lower Ol i go 4
2S 23 27 27 27 27 27
Checked Set of nested primers
This database is linked to BRCA2 gene.seq
MUNI
Restriction Enzyme Sites in Protein
File: BRCA2 gene.seq
#      EhZyme |2j4nD lZlh0D ^ISOD ^aODD tZAhOD fiSOD !^ODD ^«DD ,24i.DD   ^-1 tLÚ j^SGDD ,2SJDD ,2SĚ.DD ^SiDO |2bOD0 ^DJDO   r':        ,2ti,D0 ,2kSDD ,
33 EcoRI
34
EcoRV
33
EsP3l
36 Fsel
37 Fspl 3S IgsuI
39 Hindlll
40 Hpal
Kpnl
42 Mul
43 Muni
44 Nael
45 Narl
46 Ncol
47 Ndel
#	Enzyme	Site	#Guts	Positions & Fragment Sizes												
																
4L	Kpnl	GT2VpzY6	S	-21253	23654	68	23722	52	23774	237	24011	585	24596	L62	24758	
				629	25387	1219	26606	22851								J
42	Mlul	TR L RVyA 7	5	-22233	22674	2824	25498	576	26074	106	26L80	244	26424	23033		
43	Muni	QL3Nawl5	L0	-212S7	23620	355	23975	351	24326	282	24608	242	24850	72	24922	
				351	25273	714	25987	187	26L74	420	26594	22863				
44	Nael	AC2PAxR6	7	-21S23	230B4	597	236S1	1286	24967	86	25053	573	25626	L49	25775	
				623	26398	23059										
45	Narl	CA2APZR6	L	-20043	24S64	24593										f.
46	Ncol	PW3HGwM5	4	-22361	22546	336	22882	887	23769	531	24300	25157				
47	Ndel	HM2lawY5	2	-20366	24541	1211	25752	23705								
4S	Nhcl	A52LAK-6	L6	-22276	22631	322	22953	LS5	2313S	88	23226	27	21211	461	23714	
				369	240S3	312	24395	288	24683	151	24834	273	25L07	536	25643	1
				402	26045	30	26075	210	26285	372	26657	22800				T
	■i ■ ■				-i -i J 1- r.		-.-./--I r.		-í-ií- A r.	i A r-		■nr. A	-.-i r r.r.	w-nr-	-i A r.nr-	
Search: 22454 to 27004 End Cut Type: Blunt. Qdd, 3-overriang. S'-overhang
O O O Hybridization Time
file: M13MP1S
DNA Length: Concentration:
21
nt.
200.0 nM
L.298 MQ/mL
TO = 45.4 sec T   =3 min 47 sec
I
eo
Concentrations
i
fi e: BRCA2 gene.seq
© Constant Concentration     Q Constant Volume
© Current + Oligo: (^) Current +Oligo: C Entire Sequence (ds): (^) Forward Primer: C Reverse Primer: 0 PCR Product (ds):
Upper Oligo: Q Lower Oligo:
5.OS nmol/OD, 32.5 pg/OD 4.67 nmol/ODr 30.9 pg/OD 0.001 nmol/ODh4S.L pg/OD 5.9S nmol/ODr35.0 pg/OD 5.31 nmol/00,34.0 pg/OD 0.146 nmol/ODr 4S.L pg/OD 4.S3 nmol/ODr31.2 pg/OD 4.67 nmol/ODr 30.9 pg/OD
32.5
MS
or
or
in
L.O OD(260)
5.0S4 nmol
50S.4 pL
yields
LO.O pM
MUNI
AHP2 cDNA (TAIR database)!
Sequence: AT3G29350.1 Date last modified   2007-04-17 Name   AT3G29350.1 Tail Accession   Sequence:40 10737427 Sequence Length (lip) 827
1 ACAATTCGCG AGAAAGACAA AACACAAGTT TCTTCTTCTT GGGATTGGCT 51 ATTTCCAGAA ATCCAAGTCA ATAATCAAAG TCCAAACAAA AAAATCCTCT 101 CCCAATCTCC GCTTCACTCT TCTCATGGAC GCTCTCATTG CTCAGCTTCA 151 GAGACAATTT CGTGATTACA CCATTTCTCT CTACCAACAG GGGTTTTTGG 201 ATGATCAATT TACTGAGTTG AAAAAGCTAC AAGATGATGG AAGTCCTGAT 251 TTTGTGTCTG AAGTGCTTTC ACTTTTCTTT GAAGATTGTG TGAAGCTTAT 301 CAGTAACATG GCTAGAGCTT TGGACACGAC AGGAACTGTA GATTTTAGTC 351 AGGTAGGTGC TAGTGTGCAT CAATTGAAGG GTAGTAGCTC AAGTGTTGGT 401 GCCAAGAGGG TCAAAACTTT GTGTGTTAGC TTCAAGGAAT GTTGTGAAGC 451 TAAGAACTAC GAAGGGTGTG TGAGATGTTT GCAGCAAGTG GATATTGAGT 501 ACAAGGCGTT AAAGACAAAG CTTCAAGATA TGTTCAATCT TGAGAAACAG 551 ATCATTCAAG CTGGTGGTAT AGTTCCTCAA GTGGATATTA AC T AAA GAGA 601 CTAGTCCATA AGAAGAAAAA AG AT GATGAC TTTCTTTCTT TAGTTTCTCT 651 TCTAAATTAT TTTGGATTTG GTGTTTGCTC AAAAACTCAA TAAAATATGT 701 GCAAAAAGAA ACAAAAACAA GTGATGGTTG TTTATAAATC AGTAGTATGT 751 ATTGTTTGAT CTCATCCGAG AAAATTGAAA CCATTGGACT AATGAATGTG 801 ATGATAATAT ATATTGGTTT GCTTCTG
MUNI
101 CCCAATCTCC GCTTCACTCT TCTCATGGAC GCTCTCATTG CTCAGCTTCA 151 GAGACAATTT CGTGATTACA CCATTTCTCT CTACCAACAG GGGTTTTTGG 201 ATGATCAATT TACTGAGTTG AAAAAGCTAC AAGATGATGG AAGTCCTGAT 251 TTTGTGTCTG AAGTGCTTTC ACTTTTCTTT GAAGATTGTG TGAAGC TT AT 301 CAGTAACATG GCTAGAGCTT TGGAC AC GAC AGGAACTGTA GATTTTAGTC 351 AGGTAGGTGC TAGTGTGCAT CAATTGAAGG GTAGTAGCTC AAGTGTTGGT 401 GCCAAGAGGG TCAAAACTTT GTGTGTTAGC TTCAAGGAAT GTTGTGAAGC 451 TAAGAACTAC GAAGGGTGTG TGAGATGTTT GCAGCAAGTG GATATTGAGT 501 ACAAGGCGTT AAA GAG A AAG CTTCAAGATA TGTTCAATCT TGAGAAACAC 551 ATCATTCAAG CTGGTGGTAT AGTTCCTCAA GTGGATATTA ACT AAA GAGA
EcoRI restriction site
5'......GlAATTC......3
3"......CTTAAI G......5'
Design of primers
AHP2ex_up
y- CCG GAA TTC ATG GAG GCT CTC ATT GCT CAG - 3'
AHP2ex_low
53- CCG GAA TTC TTA GTT A AT ATC CAC TTG AGG - 3'
I
10:
1 CCCAATCTCC GCTTCACTCT TCTCgTCGgT GCTCTCATTG CTCAqCTTCA
151 GAGACAATTT CGTGATTACA CCATTTCTCT CTACCAACAG GGGTTTTTGG 201 ATGATCAATT TACTGAGTTG AAAAAGCTAC AAGATGATGG AAGTCCTGAT 251 TTTGTGTCTG AAGTGCTTTC ACTTTTCTTT GAAGATTGTG TGAAGCTTAT 301 CAGTAACATG GCTAGAGCTT TGGACACGAC AGGAACTGTA GATTTTAGTC 351 AGGTAGGTGC TAGTGTGCAT CAATTGAAGG GTAGTAGCTC AAGTGTTGGT 401 GCCAAGAGGG TCAAAACTTT GTGTGTTAGC TTCAAGGAAT GTTGTGAAGC 451 TAAGAACTAC GAAGGGTGTG TGAGATGTTT GCAGCAAGTG GATATTGAGT 501 ACAAGGCGTT AAAGACAAAG CTTCAAGATA TGTTCAATCT TGAGAAACAG 551 ATCATTCAAG CTGGTGGTAT AGTTpiHTOA4 (.ITGGATATTA ACTA^AGAGA
EcoRI restriction site
5'......G| AATTC......3
3"......CTTAAl G......5'
Design of primers
AHP2ex_up
53- CCG GAA TTC ATG GAC GCT CTC ATT GCT CAG - 33
AHP2ex_low
5'- CCG GAA TTC TTA GTT AAT ATC CAC TTG AGG - V
U l\l I Additional resources for interested
Computational and Structural Biotechnology Journal 17, 2019,1056-1065. In-silico Design
of DNA Oligonucleotides: Challenges and Approaches, includes a list ofoligo design tools
https://www.sciencedirect.com/science/article/pii/S2001037019300844
www.protocol-online.org/prot/Research Tools/Online Tools/Oligo Design/index.html
OLIGO 7 Power Point Presentation http://oligo.net/tutorials.html
OLIGO Primer analysis software, Version 7 http://oligo.net/tutorials.html
https://langdalelab.files.wordpress.com/2015/07/degenerate-primer-design.pdf
http://www.genomecompiler.com/tips-for-efficient-primer-design/ (2015)
Nature Methods 2014, 11 (5): 499. Large-scale de novo DNA synthesis: technologies and applications
Expert Opin Ther Pat. 2014, 24(7):801-19. Oligonucleotide delivery: a patent review (2010 - 2013). Laboratory Methods in Enzymology 529: DNA Explanatory Chapter: PCR Primer Design (2013) PCR Primer Design; IMBB Workshop, N. Ndegwa (2013) PCR Primer Design; A. Yuryev (2010)
AAPS Journal 2009, 11(1): 195 - 203. Targeted Delivery Systems for Oligonucleotide Therapeutics
iCODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer)PCR primer design (2009) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2703993/
Bioinformatic tools and guideline for PCR primer design. Kamel A. Abd-Elsalam (2003) Artificial DNA: Methods and Applications; Khudyakov, Y.E., Fields, W.A., Ed. (2003) PCR Primer: A Laboratory Manual (2003)
III
Discovery is not in seeking new landscapes, but in having new eyes...
Marcel Proust