Chapter 1

A Guide to Using STITCHER for Overlapping Assembly

PCR Applications
Damien M. O’Halloran

Abstract
Overlapping PCR is commonly used in many molecular applications that include stitching PCR fragments
together, generating fluorescent transcriptional and translational fusions, inserting mutations, making
deletions, and PCR cloning. Overlapping PCR is also used for genotyping and in detection experiments
using techniques such as loop-mediated isothermal amplification (LAMP). STITCHER is a web tool pro-
viding a central resource for researchers conducting all types of overlapping assembly PCR experiments
with an intuitive interface for automated primer design that’s fast, easy to use, and freely available online.

Key words PCR, Overlapping PCR, Assembly PCR, Primer design, PCR fusion, Molecular biology

1 Introduction

Overlapping PCR can be used for a wide number of experiments,

including “stitching” PCR fragments together, generating GFP/
RFP transcriptional and translational fusions, inserting mutations,
making deletion mutations, and Loop-mediated isothermal
AMPlification (LAMP) [1–5]. STITCHER is a web-based tool
(Fig. 1), which aims to provide researchers conducting all types of
overlapping PCR applications with a fast, easy to use, automated
method for returning all necessary primers [6]. STITCHER can
handle both single sequence input and multisequence input.
STITCHER can be used to provide primers for removing sections
of DNA using the “deletion mutation” feature or inserting muta-
tions using the “user-defined overlapping sequence” feature.
STITCHER can also search for off-target sites for selected primers
on each strand for specific species.

Randall A. Hughes (ed.), Synthetic DNA: Methods and Protocols, Methods in Molecular Biology, vol. 1472,
DOI 10.1007/978-1-4939-6343-0_1, © Springer Science+Business Media New York 2017

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4 Damien M. O’Halloran

stitcher | fetch | off-target | blast-off | cross-comp | articles | help

STITCHER: a web resource for high-throughput design

of primers for overlapping PCR applications

Enter any number of sequences in FASTA format:

Primer size:
Max: Min:

5' and 3' search areas (bp):

5': 3':

Primer GC %:
Upper GC: Lower GC:

Primer Tm:
Upper Tm: Lower Tm:

Salt concentration (mM):

10 50

Increment forward:
Steps (bps):

Increment reverse:
Steps (bps):

GC clamp:
Yes No

Self complementarity stringency:

Regular High None

Exclude repetitive sequence:

Yes No

Overlapping fragment:
none

User-defined overlapping reverse sequence:

User-defined overlapping forward sequence:

Deletion Mutation:
Yes No

Fig. 1 Screenshot from the STITCHER home page. Default settings are inserted for all parameters and also a
sample sequence is provided in FASTA format. The navigation menu at the top of the page can be used to
obtain sequences from NCBI (fetch), search for off-target matches (off-target and blast-off), examine cross
complementary between primers (cross-comp), read about overlapping PCR technology (articles), and learn
more about STITCHER’s features and output (help)
 Overlapping Assembly PCR 5

2 Materials

STITCHER can be found on the Internet at http://ohalloranlab.

net/STITCHER.html (Fig. 1). STITCHER was written using
Perl, CSS, JavaScript, and HTML. STITCHER has been tested on
PC and MAC, as well as iPhone and Android mobile devices, using
the following browsers: Safari, Chrome, Internet Explorer, Firefox,
and Atomic. If STITCHER does not recognize your browser, it will
return a warning. STITCHER is free to use without login require-
ments, and therefore the end user only needs a computer with
Internet connectivity and one of the browsers listed above to use it.

3 Methods

3.1 STITCHER 1. All input sequences for STITCHER must be in FASTA format
Parameters [7]. FASTA format starts with a “>” character followed by a
definition, all on the same line. STITCHER is not case sensitive,
3.1.1 Input and the DNA sequence begins on the next line for example:
> gene_1 name
ATGGTAGTATCAGCTAGCATCAGTTAG
2. The upper and lower range for primer size is provided as whole
integers (see Note 1).

3.1.2 Search Area 1. STITCHER only searches for primers within the user specified
5′ and 3′ search areas (Fig. 2).
2. STITCHER will loop over the specified search area and return
primers that match all other criteria that sit in the primer length
range such that lower length ≤ primer ≤ upper length.
3. A user setting the 5′ search to 200 bps will result in STITCHER
searching for primers within the first 200 bps. If the 5′ or 3′
search areas are greater than the size of the input DNA
sequence, this will result in a failed program execution. The 5′
and 3′ search area can be of different sizes, and including this
feature allows the user to determine the product range, which
is also returned in the output HTML file.

5’ search area
DNA sequence

3’ search area

Fig. 2 STITCHER only searches for primers within the user designated 5′ and 3′
search areas. The blue line indicates the DNA sequence
6 Damien M. O’Halloran

4. If the user selects “Deletion Mutation,” the 5′ and 3′ search

areas become the fixed nucleotide number for the deletion
area. Following the example above where the user sets the 5′
and 3′ search areas to 200 bps; if the user also selects “deletion
mutation,” STITCHER will only return one primer pair start-
ing at 200 bps in the case of the forward primer that contains
a reverse complement overlap to the reverse primer overlap,
and starting at 200 bps before the end of the input sequence
the reverse primer will be generated, assuming that all the
selected criteria can be met.

3.1.3 Primer GC% 1. Forward and reverse primer pairs should have similar Tm values
and Tm Calculation and similar GC% values. The GC% is calculated by STITCHER
by counting the number of G and C nucleotides and dividing
by the total number of nucleotides (A, C, G, T) and multiply-
ing this number by 100 to get a percentage.
2. Undetermined base pairs can be included using the letter “N”.
In the case of the reverse primer that contains either a pre-
defined overlap (e.g., GFP or mCherry) or a user-defined over-
lap: the GC% is calculated only from the designed portion (i.e.,
not the overlap—this is also the case for primer Tm values) (see
Notes 2 and 3).
3. The default formula used to calculate the primer melting tem-
perature (Tm) assumes that the reaction is carried out in the
presence of 50 mM monovalent cations (this can be changed to
reflect reactions at 10 mM) using the nearest neighbor thermo-
dynamic calculations as follows for primers less than 36 bases:

Tm = DH / ( DS + R ´ ln ( c / 4 ) ) - 273.15 + 16.6 log éë K + ùû

where ΔH (enthalpic) and ΔS (entropic) are as described in [8, 9]

and R is the molar gas constant, and c is the total molar concentra-
tion of the annealing oligonucleotides when not self‐complemen-
tary. For primers greater than 36 bases the following formula is used:

( )
Tm = 81.5 °C + 16.6 °C x log 10  Na +  +  K +  + 0.41 °C x ( %GC) 675 / N

where N is primer length.

3.1.4 Increments 1. STITCHER moves along the input DNA in set increments
Along the Search Area (bps) determined by the user. The default step is 2 bps.
­Lowering the step increment increases the number of primers
returned. However, in several instances (e.g., Primer walking
or LAMP), defined increments are preferred to return possible
primer sequences. Therefore, the increment step can be set by
the user by entering the number representing nucleotide
counts along the input sequence.
 Overlapping Assembly PCR 7

3.1.5 Self-­ 1. Intramolecular primer interactions can occur if the primer

Complementarity exhibits excessive self-complementarity. The level of excess is
and Repetitive Sequence determined by setting a stringency control to “Regular”
(default), “High,” or “None.”
2. STITCHER can calculate three self-complementarity scores: one
score for the forward primer; one score for the designed reverse
primer; and one score for the designed reverse primer/overlap
chimera. Determining the “Regular” stringency score was done
in silico by examining the number of primers returned from ran-
dom input sequences, and also experimentally by testing returned
primers of “regular” stringency scores to ensure robust PCR
products were generated. The “High” stringency score will sig-
nificantly reduce the number of primers returned (see Note 4).
3. STITCHER’s default mode is to exclude repetitive sequence,
and in doing so STITCHER will void primer sequences that
contain >4 identical base pairs, e.g., “AAAAA” or “GGGGG”
(4 bp homopolymeric repeats are okay).
4. By excluding repetitive sequence, STITCHER will also
select against any sequence that contains four or more
dinucleotide repeats (e.g., ATATATAT or GCGCGCGC).
These repetitive sequences will significantly increase intra-
molecular interactions; however, in the case of repetitive
sequence inputs this feature can be de-selected.

3.1.6 Overlap Fragment 1. Using the drop-down selection feature the user can select a
predefined overlapping fragment (Fig. 3). Currently these
include sequences for:
GFP: ACAGCTCCTCGCCCTTGCTCACCAT
mCherry: TATCTTCTTCACCCTTTGAGACCAT
RFP: TATCTTCTTCACCCTTTGAGACCAT
YFP: ACAGCTCCTCGCCCTTGCTCACCAT
tdTomato: TGACCTCCTCGCCCTTGCTCACCAT
GFP_C_elegans: AGTCGACCTGCAGGCATGCAAGCT
Illumina Paired End Adapter1:ACACTCTTTCCCTACAC
GACGCTCTTCCGATCT
Illumin Paired End Adapter 2CTCGGCATTCCTGCTGAAC
CGCTCTTCCGATCT
Illumina Paired End PCR Primer 1: A
­ ATGATACGGCGACCAC
CGAGATCTACACTCTTTCCCTACACGACGCT
CTTCCGATCT
Illumina Paired End PCR Primer 2: CAAGCAGAAGACG
G C ATA C G A G AT C G G T C T C G G C AT T C C T G C T
GAACCGCTCTTCCGATCT
Illumina Paired End Sequencing Primer 1:
8 Damien M. O’Halloran

ACACTCTTTCCCTACACGACGCTCTTCCGATCT
Illumina Paired End Sequencing Primer 2:
C G G T C T C G G C AT T C C TA C T G A A C C G C T C T T C C
GATCT
2. The overlap sequences can be used to generate transcriptional
or translational reporters fused to various fluorophores; it can
also be used in Illumina paired end sequencing reactions. In
the case of “GFP_Celegans,” the sequence is a commonly used
overlap to stitch “GFP” to a gene or promoter of interest (see
[5, 10] for more details) (see Note 5).
3. As per the “user-defined reverse overlap” textbox, the “user-­
defined forward overlap” textbox can be used to add any
sequence to the 5′ end of the forward primer. In most cases,
users will want to design primers with a specific overlap to their
input DNA. In these cases, the user can input a DNA sequence
in the “forward” or “reverse” (or both) overlap sequence text-
boxes and the primers that are returned will contain the user
specified overlap sequence of any length.

3.1.7 Deletion Overlap 1. Overlapping PCR can be used to delete segments of DNA
(Fig. 3). In this case the user will usually have a specific area in
mind to be deleted and therefore using 5′ and 3′ search areas
will not make sense. If the user selects the “deletion mutation”
feature the values entered in the 5′ and 3′ search area text boxes
will be used to generate a single primer pair that starts from the
base pair entered in the 5′ and 3′ search area text boxes.
2. By selecting the “deletion mutation” feature the forward
primer will automatically contain a reverse complement match
at its 5′ end to the reverse primer’s overlapping segment; thus,
the user only needs to enter a sequence for the “reverse over-
lap” and not the “forward overlap.”

3.2 Remotely STITCHER also includes a tool called “fetch” that will return
Retrieving Sequences sequences from NCBI using Entrez Programming Utilities [www.
ncbi.nlm.nih.gov/books/NBK25501/]. To use fetch, the user
inputs one or more accession numbers into the textbox, and after
clicking “submit,” the program returns the nucleotide sequences
in FASTA format by printing to the browser.

3.3 Off-Target 1. Once a primer of interest is selected using the features outlined
Detection above, it can then be screened for off-target hits using
STITCHER’s “off-target detection” feature (see Note 6).
2. The user can enter the number of mismatches permitted in the
off-target search. An integer must be entered in this box. Use
zero for no mismatches. Also, if this feature is selected
STITCHER can examine both strands of DNA for potential
 Overlapping Assembly PCR 9

Fig. 3 Overview of the main STITCHER facilitated PCR applications. In the left column the cartoon indicates
input type and the desired applications, which are then represented in cartoon form in the middle column. The
column on the right indicates the STITCHER specific features that enable each application

off-target matches incorporating in each case the number of

mismatches entered in the mismatch textbox.

3.4 Remote BLAST The user can remotely perform BLAST searches using the “blast‐
Searches off” tool to search for off‐target hits against NCBI’s nucleotide
non‐redundant database. Output from the BLAST search is printed
to the browser. This facility can be useful to search for nonspecific
primer matches.

3.5 Checking Cross The cross complementarity tool allows the user to input forward
Complementarity and reverse primers, and calculates the free energy (ΔG°kj/mol) of
DNA duplexes using the formula:

DG  ( total ) = DH total - T DS total

10 Damien M. O’Halloran

where enthalpic, ΔH°, and entropic, ΔS°, parameters are as

described in [11]. The cross complementarity tool also returns a
warning if significant complementarity exists between each primer,
where significance was tuned based upon Primer3Plus [12] com-
plementarity values from 100 primer pairs. Users can input the
selected primer pairs and test for significant primer dimer using
STITCHER’s cross complementarity tool.

3.6 Literature Search The end user can also stay up to date with recent articles related to
on Overlapping PCR overlapping PCR technology through STITCHER’s “articles”
Applications page. Clicking “get papers” or “get more papers” will return
matches for articles related to overlapping PCR.

4 Notes

1. Usually, primer lengths from 18 base pairs to 28 bps work well

for routine PCR conditions.
2. The presence of a GC dinucleotide combination at the 3′ end
of the primer facilitates specific binding on account of the
greater strength in bonds between G and C base pairs. If this
feature is selected, STITCHER will only return primers where
a suitable GC dinucleotide combination at the 3′ end of the
forward primer can be detected.
3. Usually, a GC content between 40 and 60 % is appropriate.
4. Setting the score to “None” can be very useful where highly
repetitive sequence is examined.
5. The user can also select none of the predefined overlapping
fragments, and instead primers without overlaps can be gener-
ated for the ligation PCR step, or for traditional PCR. If the
user does not want to use one of the predefined reverse overlap
fragments, it is also possible to input any sequence into the
“user-defined overlapping sequence” textbox and instead this
sequence will be incorporated into the program.
6. As of now this function only permits screening against the fol-
lowing genomes:
Tannerella_forsythia ATCC_43037_uid83157/NC_016610
Staphylococcus_aureus_MRSA252 uid57839/NC_002952
Candidatus_Liberibacter americanus_Sao_Paulo_
uid227424/NC_022793
Bacillus anthracis CDC_684_uid59303/NC_012577
Bordetella pertussis CS_uid158859/NC_017223
Plasmodium falciparum 3D7 version 2.1.5
Streptococcus pneumoniae ATCC_700669
 Overlapping Assembly PCR 11

Mycobacterium tuberculosis Erdman_ATCC_35801

Escherichia_coli K12_substr_W3110_uid161931
Klebsiella_pneumoniae 1084_uid174151/NC_018522

Acknowledgements

Support for this work was provided by The George Washington

University (GWU) Columbian College of Arts and Sciences, GWU
Office of the Vice President for Research, and the GWU Department
of Biological Sciences.

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