k.) 1990 Oxford University Press
6460 Nucleic Acids Research, Vol. 18, No. 21
DNA polymers of protein binding sequences generated by PCR William A.Rudert and Massimo Trucco* Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA 15213-2583, USA Submitted September 19, 1990 Gene expression is regulated by trans-acting factors that bind to highly conserved nucleotide sequences present in the promoter region. Frequently, during the study of these processes, oligomers of the recognition sites of these DNA binding nuclear proteins have been constructed by ligation of annealed complementary synthetic oligonucleotides (1). This approach involves the production of large quantities of oligonucleotides and generates possibly unstable polymers. In another approach, the construct's stability in a host plasmid was increased by repetitive head to tail directed cloning of the monomeric recognition sequence (2). This method is quite time consuming. This report describes a different approach to generate multiple head to tail tandem sequences using the polymerase chain reaction (PCR) on staggered complementary oligonucleotides which function as both primers and templates. Our approach is simple, reliable, relatively inexpensive and quite rapid. As an example, figure 1 shows the sequences which were used as primers to create tandem repeats of a methylation protected region of the X-conserved box of the HLA-DQ,B gene promoter (3). The primers must be selected so that the first is centered on the recognition site itself while the second is centered on the junction of the tail of one recognition sequence and the head of the next adjacent one. A few more nucleotides may be added at the oligomer junctions, to create a restriction site between the multiple recognition sequences of the polymers. In our example, TATC was added at both the 3' and 5' ends of the recognition sequence to create EcoRV sites (GATATC). The 3' ends of the primers must be carefully selected to avoid undesirable primer artifacts. For example, in the particular case shown here, the stretches of A's and T's were deliberately positioned near the centers of the primers rather than at the 3' ends, by extending the primers' length a few bases (e.g., CTAGA). The sizes of our synthetic oligonucleotides were thus increased to 31 and 28 bases, respectively. These oligonucleotides were useful in generating polymers with 22 nucleotide long repeats. As illustrated, the early rounds of amplification generate 44 base pair products which create the basis for a different, staggered annealing during the later cycles which then leads to the progressive increase of the polymer size. The amplification in the example, was performed in two steps. In the first, 40 cycles of denaturation at 900C for 1 min, annealing at 440C for 1.5 min, and elongation at 72°C for 1.5 min were used with a beginning primer concentration of 3 1M. The material from three tubes was pooled and then separated in low melting point agarose (1.7% in TAE buffer). The predominant bands, which *
To whom correspondence should be addressed
corresponded to 4 and 5 repeat polymers, were isolated, eluted, and concentrated. They were then used as primers and templates for 25 additional cycles of amplification. This product was concentrated and washed in a Centricon device (Amicon), phosphorylated using T4 polynucleotide kinase, ligated into the EcoRV site of pBluescript II, and cloned in XLl-Blue (Stratagene). Figure 2A shows the size range of the cloned inserts which corresponded to 11-70 repeat polymers. Figure 2B shows the digestion of each insert with EcoRV. In the example, the conditions used were such as to only partially digest the larger inserts. Polymeric sequences generated in this way may be used to prepare DNA binding protein affinity columns, and as probes which can be used for the screening of expression libraries in search of the genes encoding these DNA binding proteins.
REFERENCES 1. Kadonaga,J.T. and Tjian,R. (1986) Proc. Natl. Acad. Sci. USA 83, 5889-5893. 2. Eisenberg,S., Francesconi,S.C., Civalier,C. and Walker,S.S. (1990) Methods Enzymol. 182, 521-529. 3. Turco,E., Manfras,B.J., Ge,L., Rudert,W.A. and Trucco,M. (1990) Immunogenetics In press. .....
AAAMA
GA.
Protein Binding Region
cTAaaT.aM!yCec!AMcAAATGTCTGC
A
First Cycle of PCR
A Stggered Annealing GATCTATAGTTTTTAGACGGCTh2AGTI¶WAC&ACG
C~&~AAMQZCTcCZA~MAAAAATGTCTGC A
EcoRV
A
A
Figure 1. The protein binding region of the HLA DQ,B gene promoter is underlined. The oligonucleotides used as both primers and templates are in bold. Triangles indicate the EcoRV restriction sites.
Figure 2. A)
Polynieric
inserts were cut from the vector
and Hindil, and separated in a
1.6 % agarose gel.
DNA Ladder
(BRL). B) EcoRV digestion of the
The
were
products
separated in
a
12 %
by
digestion with EcoRi
In the first lane is the !Kb same
acrylamide
gel.
plasmids
shown in A.
6460 Nucleic Acids Research, Vol. 18, No. 21
DNA polymers of protein binding sequences generated by PCR William A.Rudert and Massimo Trucco* Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA 15213-2583, USA Submitted September 19, 1990 Gene expression is regulated by trans-acting factors that bind to highly conserved nucleotide sequences present in the promoter region. Frequently, during the study of these processes, oligomers of the recognition sites of these DNA binding nuclear proteins have been constructed by ligation of annealed complementary synthetic oligonucleotides (1). This approach involves the production of large quantities of oligonucleotides and generates possibly unstable polymers. In another approach, the construct's stability in a host plasmid was increased by repetitive head to tail directed cloning of the monomeric recognition sequence (2). This method is quite time consuming. This report describes a different approach to generate multiple head to tail tandem sequences using the polymerase chain reaction (PCR) on staggered complementary oligonucleotides which function as both primers and templates. Our approach is simple, reliable, relatively inexpensive and quite rapid. As an example, figure 1 shows the sequences which were used as primers to create tandem repeats of a methylation protected region of the X-conserved box of the HLA-DQ,B gene promoter (3). The primers must be selected so that the first is centered on the recognition site itself while the second is centered on the junction of the tail of one recognition sequence and the head of the next adjacent one. A few more nucleotides may be added at the oligomer junctions, to create a restriction site between the multiple recognition sequences of the polymers. In our example, TATC was added at both the 3' and 5' ends of the recognition sequence to create EcoRV sites (GATATC). The 3' ends of the primers must be carefully selected to avoid undesirable primer artifacts. For example, in the particular case shown here, the stretches of A's and T's were deliberately positioned near the centers of the primers rather than at the 3' ends, by extending the primers' length a few bases (e.g., CTAGA). The sizes of our synthetic oligonucleotides were thus increased to 31 and 28 bases, respectively. These oligonucleotides were useful in generating polymers with 22 nucleotide long repeats. As illustrated, the early rounds of amplification generate 44 base pair products which create the basis for a different, staggered annealing during the later cycles which then leads to the progressive increase of the polymer size. The amplification in the example, was performed in two steps. In the first, 40 cycles of denaturation at 900C for 1 min, annealing at 440C for 1.5 min, and elongation at 72°C for 1.5 min were used with a beginning primer concentration of 3 1M. The material from three tubes was pooled and then separated in low melting point agarose (1.7% in TAE buffer). The predominant bands, which *
To whom correspondence should be addressed
corresponded to 4 and 5 repeat polymers, were isolated, eluted, and concentrated. They were then used as primers and templates for 25 additional cycles of amplification. This product was concentrated and washed in a Centricon device (Amicon), phosphorylated using T4 polynucleotide kinase, ligated into the EcoRV site of pBluescript II, and cloned in XLl-Blue (Stratagene). Figure 2A shows the size range of the cloned inserts which corresponded to 11-70 repeat polymers. Figure 2B shows the digestion of each insert with EcoRV. In the example, the conditions used were such as to only partially digest the larger inserts. Polymeric sequences generated in this way may be used to prepare DNA binding protein affinity columns, and as probes which can be used for the screening of expression libraries in search of the genes encoding these DNA binding proteins.
REFERENCES 1. Kadonaga,J.T. and Tjian,R. (1986) Proc. Natl. Acad. Sci. USA 83, 5889-5893. 2. Eisenberg,S., Francesconi,S.C., Civalier,C. and Walker,S.S. (1990) Methods Enzymol. 182, 521-529. 3. Turco,E., Manfras,B.J., Ge,L., Rudert,W.A. and Trucco,M. (1990) Immunogenetics In press. .....
AAAMA
GA.
Protein Binding Region
cTAaaT.aM!yCec!AMcAAATGTCTGC
A
First Cycle of PCR
A Stggered Annealing GATCTATAGTTTTTAGACGGCTh2AGTI¶WAC&ACG
C~&~AAMQZCTcCZA~MAAAAATGTCTGC A
EcoRV
A
A
Figure 1. The protein binding region of the HLA DQ,B gene promoter is underlined. The oligonucleotides used as both primers and templates are in bold. Triangles indicate the EcoRV restriction sites.
Figure 2. A)
Polynieric
inserts were cut from the vector
and Hindil, and separated in a
1.6 % agarose gel.
DNA Ladder
(BRL). B) EcoRV digestion of the
The
were
products
separated in
a
12 %
by
digestion with EcoRi
In the first lane is the !Kb same
acrylamide
gel.
plasmids
shown in A.
