**5. Results**

## **5.1 Analysis of cloned nucleotide sequences from PCRs with primers specific for** *papA*

An annealing temperature of 55°C was used in the PCRs for the amplification of the *papA* gene with primers 22 and 23. The obtained PCR products were all of the expected size (around 600 bp). However, the nucleotide sequence analysis of the eight obtained cloned PCR products revealed that six clones harboured false, non-*papA* inserts. Four of these false clones derived from amplification of part of methylisocitrate lyase gene, and two clones derived from amplification of part of the RNA-binding protein Hfq gene and part of the GTPase HflX gene, as revealed by BLAST analysis. In both cases even though both primers, forward primer 22 and reverse primer 23, were added to the PCR mixture, the primer 22 was used as the forward but also the reverse primer. Further nucleotide analysis revealed that in the case of the amplification of part of the methylisocitrate lyase gene, the forward primer annealed downstream from the c348059 position of the 3′ → 5′ strand and reverse primer upstream of the 348539 position on the 5′ → 3′ DNA strand of the *E. coli* K-12 MG1655 sequence as deposited in the CP025268.1 nucleotide sequence [35]. The anticipated annealing sites for nonspecific *papA*-primer binding in this case are presented in **Figure 3**.

In the case of the amplification of part of the RNA-binding protein Hfq gene and part of the GTPase HflX gene, the forward primer annealed downstream from

**173**

**Figure 4.**

**Figure 3.**

*347733 to 348623 nt.*

presented in **Figure 4**.

*is positioned from 4402598 to 4403878 nt.*

*Annealing Temperature of 55°C and Specificity of Primer Binding in PCR Reactions*

*Anticipated annealing sites for non-specific papA-primer binding in the methylisocitrate lyase gene. The shown sequences are enumerated according to the CP025268.1 GenBank deposited sequence [35]. The sequence* 

*the sequence from 348033 to 348059 nt is shown, and for the reverse primer annealing site, the sequence from 348539 to 348565 nt is shown. The primer sequence is in the grey box. The arrows mark the direction of DNA elongation in the PCR. The methylisocitrate lyase gene is positioned in the deposited sequence from* 

*and the complement chromosomal sequence are given. For the forward primer annealing site,* 

the c4402446 position on the 3′ → 5′ DNA strand, and the reverse primer annealed upstream from the 4402903 position on the 5′ → 3′ DNA strand of the *E. coli* K-12 MG1655 sequence as deposited in the CP025268.1 nucleotide sequence [35]. The anticipated annealing sites for non-specific *papA*-primer binding in this case are

*Anticipated annealing sites for non-specific papA-primer binding in the RNA-binding protein Hfq gene (forward primer) and GTPase HflX gene (reverse primer). The shown sequences are enumerated according the CP025268.1 GenBank deposited sequence [35]. The sequence and the complement chromosomal sequence are given. For the forward primer annealing site, the sequence from 4402419 to 4402446 nt is shown, and for the reverse primer annealing site, the sequence from 4402903 to 4402934 nt is shown. The primer sequence is in the grey box. The arrows mark the direction of DNA elongation in the PCR. The RNA-binding protein Hfq gene is positioned in the deposited sequence from 4402214 to 4402522 nt, and the GTPase HflX gene* 

*DOI: http://dx.doi.org/10.5772/intechopen.85164*

#### *Annealing Temperature of 55°C and Specificity of Primer Binding in PCR Reactions DOI: http://dx.doi.org/10.5772/intechopen.85164*

#### **Figure 3.**

*Synthetic Biology - New Interdisciplinary Science*

according to the manufacturers' protocols.

**PCRs for P- and F17-fimbrial genes**

searching for homology in the GenBank nr database.

1% of agarose gels with 0.5 μg/ml ethidium bromide, run in 0.5 × TBE electrophoresis buffer. Samples of obtained PCR products (25 μl of PCR products, 5 μl of 6 × loading dye) were subjected to analysis with agarose gel electrophoresis using 1% of agarose gels with 0.5 μg/ml ethidium bromide, run in 1 × TAE electrophoresis buffer. Used protocols for agarose gel electrophoresis were based on Sambrook et al. [34]. For DNA ladder the lambda bacteriophage DNA digested with the restriction endonuclease *Pst*I was used.

**4.6 Cloning of PCR products and DNA sequencing of cloned PCR products** 

Cloning of PCR products and DNA sequencing of cloned PCR products obtained in the PCRs for P- and F17-fimbrial genes was done as described by Starčič et al. [5]. In short, obtained PCR products were cut out of the agarose gel, cleaned with the GeneClean II Kit, inserted into the TA cloning vector pMOSBlue and then transformed to electrocompetent *E. coli* pMOSBlue cells. Subsequently, the plasmid DNA was isolated from pMOSBlue cells using the FlexiPrep Kit, and the nucleotide sequence was determined with the dideoxynucleotide chain termination method using an automated laser fluorescence sequencer. All procedures were performed

**4.7 Analysis of cloned nucleotide sequences of the PCR products amplified in** 

Sequence analysis of the cloned fragments, originated from PCR products obtained in PCRs for P- and F17-fimbrial genes, was performed with the computer program BLAST on the Internet page of the National Center for Biotechnology Information, US National Library of Medicine (http://www.ncbi.nlm.nih.gov)

**5.1 Analysis of cloned nucleotide sequences from PCRs with primers** 

specific *papA*-primer binding in this case are presented in **Figure 3**.

An annealing temperature of 55°C was used in the PCRs for the amplification of the *papA* gene with primers 22 and 23. The obtained PCR products were all of the expected size (around 600 bp). However, the nucleotide sequence analysis of the eight obtained cloned PCR products revealed that six clones harboured false, non-*papA* inserts. Four of these false clones derived from amplification of part of methylisocitrate lyase gene, and two clones derived from amplification of part of the RNA-binding protein Hfq gene and part of the GTPase HflX gene, as revealed by BLAST analysis. In both cases even though both primers, forward primer 22 and reverse primer 23, were added to the PCR mixture, the primer 22 was used as the forward but also the reverse primer. Further nucleotide analysis revealed that in the case of the amplification of part of the methylisocitrate lyase gene, the forward primer annealed downstream from the c348059 position of the 3′ → 5′ strand and reverse primer upstream of the 348539 position on the 5′ → 3′ DNA strand of the *E. coli* K-12 MG1655 sequence as deposited in the CP025268.1 nucleotide sequence [35]. The anticipated annealing sites for non-

In the case of the amplification of part of the RNA-binding protein Hfq gene and part of the GTPase HflX gene, the forward primer annealed downstream from

**amplified in PCRs for P- and F17-fimbrial genes**

**172**

**5. Results**

**specific for** *papA*

*Anticipated annealing sites for non-specific papA-primer binding in the methylisocitrate lyase gene. The shown sequences are enumerated according to the CP025268.1 GenBank deposited sequence [35]. The sequence and the complement chromosomal sequence are given. For the forward primer annealing site, the sequence from 348033 to 348059 nt is shown, and for the reverse primer annealing site, the sequence from 348539 to 348565 nt is shown. The primer sequence is in the grey box. The arrows mark the direction of DNA elongation in the PCR. The methylisocitrate lyase gene is positioned in the deposited sequence from 347733 to 348623 nt.*

#### **Figure 4.**

*Anticipated annealing sites for non-specific papA-primer binding in the RNA-binding protein Hfq gene (forward primer) and GTPase HflX gene (reverse primer). The shown sequences are enumerated according the CP025268.1 GenBank deposited sequence [35]. The sequence and the complement chromosomal sequence are given. For the forward primer annealing site, the sequence from 4402419 to 4402446 nt is shown, and for the reverse primer annealing site, the sequence from 4402903 to 4402934 nt is shown. The primer sequence is in the grey box. The arrows mark the direction of DNA elongation in the PCR. The RNA-binding protein Hfq gene is positioned in the deposited sequence from 4402214 to 4402522 nt, and the GTPase HflX gene is positioned from 4402598 to 4403878 nt.*

the c4402446 position on the 3′ → 5′ DNA strand, and the reverse primer annealed upstream from the 4402903 position on the 5′ → 3′ DNA strand of the *E. coli* K-12 MG1655 sequence as deposited in the CP025268.1 nucleotide sequence [35]. The anticipated annealing sites for non-specific *papA*-primer binding in this case are presented in **Figure 4**.

#### **5.2 Analysis of cloned nucleotide sequences from PCRs with primers specific for** *papEF*

In the PCRs for the *papEF* amplification, also the annealing temperature of 55°C was used. Seven PCR products, all of the expected size, of around 1400 bp, were cloned, and the obtained insert sequences were analysed. All seven clones harboured the amplified *papEF*-related sequence, the *prsEF* sequence of the Prs-fimbriae (GenBank X61238.1 [36]); however, in all seven cases, only the forward POP primer annealed to the correct complementary sequence from c27 to c48 nt on the 3′ → 5′ DNA strand of the X61238.1, while the reverse primer was not as expected the BAD primer but again the POP primer, which annealed at another partially complementary sequence of the *prsEF* gene from 1357 upstream on the 5′ → 3′ DNA strand. Further BLAST analysis showed that the BAD primer has only a partial complementary region of nine nucleotides at the position 3229 to 3237 in the 5′ → 3′ DNA strand and at the position c3238 to c3230 in the 3′ → 5′ strand of the X61238.1 sequence. The anticipated annealing sites of POP primer on the analysed X61238.1 nucleotide sequences are presented in **Figure 5**.

### **5.3 Analysis of cloned nucleotide sequences from PCRs with primers specific for** *papG*

In the PCRs for the *papG* amplification, also the annealing temperature of 55°C was used. Three PCR products, all of the expected size, of around 1000 bp, were obtained and cloned, and the obtained insert sequences were analysed. All three clones harboured the expected *papG* sequence. In all three PCR amplifications, both primers, the forward GOD1 and reverse GOD2 primer, annealed at the expected positions. The anticipated annealing sites for specific *papG*-primer binding

**175**

**Figure 7.**

*Annealing Temperature of 55°C and Specificity of Primer Binding in PCR Reactions*

of GOD1 and GOD2 as revealed by analysis of the nucleotide *E. coli* sequence

*Anticipated annealing sites for non-specific F17G-primer binding in the rtn gene. The shown sequences are enumerated according the CP025268.1 GenBank deposited sequence [35]. The sequence and the complement chromosomal sequence are given. For the forward primer, F17G-1, annealing site, the sequence from 2275306 to 2275331 nt is shown, and for the reverse primer, F17G-2, annealing site, the sequence from 2276103 to 2276129 nt is shown. The primer sequence is in the grey box. The arrows mark the direction of DNA elongation in the* 

*PCR. The rtn gene is positioned in the deposited sequence from 2274762 to 2276318 nt.*

*Anticipated annealing sites of primer GOD1 and primer GOD2 in the papG sequence. The shown sequences are enumerated according the CP027701.1 GenBank deposited sequence [37]. The sequence and the complement chromosomal sequence are given. For the forward primer annealing site, the sequence from 507724 to 507748 nt is shown, and for the reverse primer annealing site, the sequence from 508702 to 508723 nt is shown. The primer sequence is in the grey box. The arrows mark the direction of DNA elongation in the PCR. The papG gene is* 

At the annealing temperature of 55°C with primers specific for the *F17G* gene, eight PCR products, again all of the expected size of approximately 900 bp, were obtained and cloned. Nucleotide sequence analysis of all eight clones showed that

**5.4 Analysis of cloned nucleotide sequences from PCRs with primers** 

*DOI: http://dx.doi.org/10.5772/intechopen.85164*

CP027701.1 [37] are presented in **Figure 6**.

*positioned in the deposited sequence from 507716 to 508726 nt.*

**specific for** *F17G*

**Figure 6.**

**Figure 5.** *Anticipated annealing sites of primer POP in the prsEF sequence. The shown sequences are enumerated according the X61238.1 GenBank deposited sequence [36]. The sequence and the complement chromosomal sequence are given. For the forward primer annealing site, the sequence from 27 to 48 nt is shown, and for the reverse primer annealing site, the sequence from 1357 to 1375 nt is shown. The primer sequence is in the grey box. The arrows mark the direction of DNA elongation in the PCR. The prsE gene is positioned in the deposited sequence from 79 to 600 nt, and the prsF gene is positioned from 676 to 1179 nt.*

*Annealing Temperature of 55°C and Specificity of Primer Binding in PCR Reactions DOI: http://dx.doi.org/10.5772/intechopen.85164*

#### **Figure 6.**

*Synthetic Biology - New Interdisciplinary Science*

**specific for** *papEF*

**specific for** *papG*

**5.2 Analysis of cloned nucleotide sequences from PCRs with primers** 

on the analysed X61238.1 nucleotide sequences are presented in **Figure 5**.

**5.3 Analysis of cloned nucleotide sequences from PCRs with primers** 

In the PCRs for the *papG* amplification, also the annealing temperature of 55°C was used. Three PCR products, all of the expected size, of around 1000 bp, were obtained and cloned, and the obtained insert sequences were analysed. All three clones harboured the expected *papG* sequence. In all three PCR amplifications, both primers, the forward GOD1 and reverse GOD2 primer, annealed at the expected positions. The anticipated annealing sites for specific *papG*-primer binding

In the PCRs for the *papEF* amplification, also the annealing temperature of 55°C was used. Seven PCR products, all of the expected size, of around 1400 bp, were cloned, and the obtained insert sequences were analysed. All seven clones harboured the amplified *papEF*-related sequence, the *prsEF* sequence of the Prs-fimbriae (GenBank X61238.1 [36]); however, in all seven cases, only the forward POP primer annealed to the correct complementary sequence from c27 to c48 nt on the 3′ → 5′ DNA strand of the X61238.1, while the reverse primer was not as expected the BAD primer but again the POP primer, which annealed at another partially complementary sequence of the *prsEF* gene from 1357 upstream on the 5′ → 3′ DNA strand. Further BLAST analysis showed that the BAD primer has only a partial complementary region of nine nucleotides at the position 3229 to 3237 in the 5′ → 3′ DNA strand and at the position c3238 to c3230 in the 3′ → 5′ strand of the X61238.1 sequence. The anticipated annealing sites of POP primer

**174**

**Figure 5.**

*Anticipated annealing sites of primer POP in the prsEF sequence. The shown sequences are enumerated according the X61238.1 GenBank deposited sequence [36]. The sequence and the complement chromosomal sequence are given. For the forward primer annealing site, the sequence from 27 to 48 nt is shown, and for the reverse primer annealing site, the sequence from 1357 to 1375 nt is shown. The primer sequence is in the grey box. The arrows mark the direction of DNA elongation in the PCR. The prsE gene is positioned in the deposited* 

*sequence from 79 to 600 nt, and the prsF gene is positioned from 676 to 1179 nt.*

*Anticipated annealing sites of primer GOD1 and primer GOD2 in the papG sequence. The shown sequences are enumerated according the CP027701.1 GenBank deposited sequence [37]. The sequence and the complement chromosomal sequence are given. For the forward primer annealing site, the sequence from 507724 to 507748 nt is shown, and for the reverse primer annealing site, the sequence from 508702 to 508723 nt is shown. The primer sequence is in the grey box. The arrows mark the direction of DNA elongation in the PCR. The papG gene is positioned in the deposited sequence from 507716 to 508726 nt.*

of GOD1 and GOD2 as revealed by analysis of the nucleotide *E. coli* sequence CP027701.1 [37] are presented in **Figure 6**.

#### **5.4 Analysis of cloned nucleotide sequences from PCRs with primers specific for** *F17G*

At the annealing temperature of 55°C with primers specific for the *F17G* gene, eight PCR products, again all of the expected size of approximately 900 bp, were obtained and cloned. Nucleotide sequence analysis of all eight clones showed that

#### **Figure 7.**

*Anticipated annealing sites for non-specific F17G-primer binding in the rtn gene. The shown sequences are enumerated according the CP025268.1 GenBank deposited sequence [35]. The sequence and the complement chromosomal sequence are given. For the forward primer, F17G-1, annealing site, the sequence from 2275306 to 2275331 nt is shown, and for the reverse primer, F17G-2, annealing site, the sequence from 2276103 to 2276129 nt is shown. The primer sequence is in the grey box. The arrows mark the direction of DNA elongation in the PCR. The rtn gene is positioned in the deposited sequence from 2274762 to 2276318 nt.*

four harboured correct and four harboured false inserts. All four false inserts were, as BLAST revealed, sequences of the protein *rtn* gene of the *E. coli* K-12 MG1655 chromosome, as deposited in the CP025268.1 nucleotide sequence [35]. Further nucleotide analysis revealed that in the case of the *rtn* gene amplification, the forward primer F17G-1 annealed downstream from the c2275331 position on the 3′ → 5′ DNA strand and reverse primer F17G-2 upstream of the 2276103 position on the 5′ → 3′ DNA strand of the *E. coli* K-12 MG1655 CP025268.1 nucleotide sequence. The anticipated annealing sites for non-specific *F17G*-primer binding in analysed nucleotide sequences are presented in **Figure 7**.
