STEP 1
Paste the target DNA or the actual amplicon. Plain sequence or single-record FASTA; A/T/C/G only, 36–1000 nt.
Nucleic acid detection design
gDNA Designer
Provide one target DNA or amplicon and choose an Ago enzyme. The service assembles a full detection cascade from cleavage-site relationships: gDNA1, gDNA2, gDNA3 and the reporter DNA.
What is gDNA Designer
A guide-DNA design tool for Argonaute (PfAgo) nucleic acid detection: paste one target DNA or amplicon and get a complete, ready-to-order detection set — gDNA1, gDNA2, gDNA3 and the reporter DNA.
A PfAgo assay does not work with a single guide. Two guides must excise a specific fragment (gDNA4) from one strand; a third guide then cuts within that interval on the opposite strand, and the reporter DNA produces the readable signal. The four sequences constrain one another through their cleavage coordinates, so changing any one of them affects the rest.
Designing that cascade by hand is error-prone: every guide has to satisfy length, GC, homopolymer and palindrome constraints at once, while the three cut sites must stay in the right relative positions. gDNA Designer encodes those rules and returns whole cascades ranked by score from a single submission.
Each cascade comes with full coordinates — binding interval, cut position, binding strand — and a schematic that draws the cascade out, so you can sanity-check a design before committing to synthesis.
Complete set
gDNA1 / gDNA2 / gDNA3 / reporter returned together — no manual assembly.
Verifiable coordinates
Binding interval, strand and cut site for every sequence, matching the schematic.
Rules built in
Length, GC, homopolymer, palindrome and primer avoidance enforced by the engine.
Ranked cascades
Scored as whole cascades, so you can start validating from the top of the list.
How to use it
STEP 2
Enzyme is fixed to PfAgo — the only one the engine implements today, so there is nothing to choose; TtAgo is not yet available.
STEP 3
If you already have amplification primers, enter them. Primers are not design targets — they are used to avoid primer regions and to flag complementarity risk.
STEP 4
Set how many cascades to return (1–20, defaults to 20 here), then review them on the results page and check the cut relationships against the schematic.
Parameters and rules
Input parameters
Target sequence
Required. Target DNA or amplicon, A/T/C/G only, 36–1000 nt. Plain sequence or single-record FASTA (header lines are ignored).
Enzyme
Only PFAGO is supported today and the page submits it automatically; other values are rejected by the service.
Forward / reverse primer
Optional. Used only to avoid primer-covered regions and to flag guide–primer complementarity risk. Not a design target.
Result count
1–20, defaults to 20 on this page. Cascades are returned by descending total score.
Filtering rules
Guide length
Tried in priority order 16, 17, 15, 18; the gDNA4 fragment is 16, 17 or 18 nt.
GC content
Hard filter — candidates outside 40%–80% are discarded.
Homopolymers
A run of GGGG is disallowed; five or more consecutive A, C or T is also discarded.
Palindromes
A guide may not be its own reverse complement.
End complementarity
The first and last 5 nt are filtered strictly for reverse complementarity; the rule is relaxed only if strict mode yields nothing.
Output fields
gDNA1 / gDNA2
Cut the same strand at two positions; the fragment between those cuts is gDNA4.
gDNA3
Cuts the opposite strand, inside the gDNA4 interval. Whether the landing is exact is reported as gdna3_match_mode.
Reporter DNA
The reverse complement of gDNA4, producing the readable signal.
Coordinates
The API returns 0-based half-open intervals; the page shows 1-based inclusive positions and re-bases reverse-strand coordinates onto the forward strand.
Input parameters
Target sequence
Required. Target DNA or amplicon, A/T/C/G only, 36–1000 nt. Plain sequence or single-record FASTA (header lines are ignored).
Enzyme
Only PFAGO is supported today and the page submits it automatically; other values are rejected by the service.
Forward / reverse primer
Optional. Used only to avoid primer-covered regions and to flag guide–primer complementarity risk. Not a design target.
Result count
1–20, defaults to 20 on this page. Cascades are returned by descending total score.
Filtering rules
Guide length
Tried in priority order 16, 17, 15, 18; the gDNA4 fragment is 16, 17 or 18 nt.
GC content
Hard filter — candidates outside 40%–80% are discarded.
Homopolymers
A run of GGGG is disallowed; five or more consecutive A, C or T is also discarded.
Palindromes
A guide may not be its own reverse complement.
End complementarity
The first and last 5 nt are filtered strictly for reverse complementarity; the rule is relaxed only if strict mode yields nothing.
Output fields
gDNA1 / gDNA2
Cut the same strand at two positions; the fragment between those cuts is gDNA4.
gDNA3
Cuts the opposite strand, inside the gDNA4 interval. Whether the landing is exact is reported as gdna3_match_mode.
Reporter DNA
The reverse complement of gDNA4, producing the readable signal.
Coordinates
The API returns 0-based half-open intervals; the page shows 1-based inclusive positions and re-bases reverse-strand coordinates onto the forward strand.
How the design works
STEP 1
Normalise the input
01Strip FASTA headers and all whitespace, then uppercase the sequence.
02Validate alphabet and length: A/T/C/G only, 36–1000 nt.
03If primers were supplied, mark the regions they cover so candidates can avoid them.
STEP 2
Build the candidate pool
01Slide a window across the target at the enzyme’s preferred lengths to enumerate candidate guides.
02Hard-filter each candidate on GC range, GGGG runs, long A/C/T homopolymers and self-palindromes.
03Record binding interval, strand and cut position — those coordinates are what the cascade assembly depends on.
STEP 3
Assemble cascades
01Enumerate candidate gDNA4 intervals (16–18 nt); this interval determines the reporter sequence.
02Find two guides whose cut sites land exactly on the ends of that interval — these become gDNA1 and gDNA2.
03Find a guide on the opposite strand whose cut lands inside the interval — this becomes gDNA3.
STEP 4
Score and rank
01Take the reverse complement of gDNA4 as the reporter, completing the four deliverable sequences.
02Score the cascade as a whole rather than judging guides in isolation.
03Return cascades by descending total score, annotated with how gDNA3’s cut matches the gDNA4 interval.
Frequently asked questions
Why are three guides needed?
A PfAgo assay depends on a precisely excised target fragment. gDNA1 and gDNA2 create two cut sites on one strand, defining gDNA4; gDNA3 then cuts inside that interval on the opposite strand. Remove any one of them and the cascade cannot complete.
Do I need to synthesise gDNA4?
No. gDNA4 is the fragment released by cleavage, not a deliverable. It exists to explain where the reporter comes from — the reporter is its reverse complement. What you order is gDNA1, gDNA2, gDNA3 and the reporter DNA.
Should I paste the amplicon or the original gene sequence?
Paste the actual amplicon. All coordinates are relative to the sequence you submit, so using the amplicon keeps the guides inside a region that really exists in your reaction and makes primer avoidance meaningful.
What do the primer fields do?
Primers are not designed. When supplied, the engine avoids primer-covered regions and flags guides that risk hybridising with a primer, reducing non-specific interference downstream.
Why does a target sometimes return no cascades?
Usually the region is too short or its base composition is too extreme. GC must fall within 40%–80%, GGGG runs and long A/C/T homopolymers are rejected, and a guide may not be self-palindromic. Try a different region or widen the submitted sequence.
How do I read the coordinates in the results?
The API returns 0-based half-open intervals; the page converts them to 1-based inclusive positions. Coordinates given in reverse-strand numbering — gDNA3’s, typically — are re-based onto the forward strand, so all four intervals can be compared directly.
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