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CHAPTER 6

Primer Design

CHAPTER 6: Primer Design

Introduction Primers are designed to amplify a specific DNA sequence from either genomic or plasmid template DNA. For successful amplification of DNA sequences in PCR, primers need to fulfill several basic requirements: 1. The 3’ portion of each primer (about 15-20 bases) must be complementary to the DNA template. 2. The 3’ portions of primers used in PCR should not be complementary to each other, because this facilitates the formation of primer-dimers. 3. An additional sequence of bases that is noncomplementary to the template DNA may be added at the 5’ end of a primer. Extra bases are often added to primers to incorporate restriction sites into the PCR product. Restriction sites at each end of the PCR product enable future manipulations of the PCR product, such as its introduction into plasmids for gene expression.

Primer Design Each PCR requires unique primers. The nucleotide sequence of primers depends on the nucleotide sequence of the template DNA. The objective of this lab project is to amplify the ArsR gene from K12 E. coli genomic DNA using primers complementary to this gene, thus you must first obtain the DNA sequence of this gene. DNA sequences may be found in the NCBI (National Center for Biotechnology) nucleotide database at www.ncbi.nlm.nih.gov. Each sequence that has been deposited in this database has its own “ID” referred to as the accession number. If you know the accession number for a DNA sequence, you may use it to search for the desired gene, or alternatively, you may type in the name of the gene and species. The database displays the requested DNA sequence and relevant data (see website or pages 58-59). In the display you will find information on the authors and journal where the DNA
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CHAPTER 6: Primer Design

sequence was first published. The CDS (coding region) informs you of the positions of the start and stop codons in the DNA sequence. Besides bases that code for the protein, additional bases are provided as part of the sequence for most genes in the NCBI database. Some of these additional bases represent regulatory DNA sequences. According to the CDS, the adenine of the start codon (ATG) for the ArsR gene is located at position 3646551 whereas the adenine of the stop codon (TAA) is located at position 3646904 (adenine 3646904). Note that the CDS also offers the amino acid sequence of the protein encoded by this gene. Each letter represents an amino acid; the first one, encoded by ATG or start codon, is M for methionine. The base count origin (or origin) is used as a reference system for the numbering of nucleotides of the DNA sequence. Note that the published DNA sequence presents the nucleotide information for one of the stands of the DNA double helix, referred to as the sense strand. The anti-sense strand is not displayed. DNA sequences are always published from the 5’ end towards the 3’ end. Remember that each DNA strand possesses a 5’ end, which carries a phosphate group attached to the fifth carbon of the 2-deoxyribose, and a 3’ end with an OH group attached to the third carbon of the 2-deoxyribose. Two primers must be designed for PCR: the forward primer extends in PCR from the start codon towards the stop codon of template DNA, while the reverse primer extends from the stop codon towards the start codon (Figure 6.1). Thus the positions of the start and stop codons of the template DNA are important landmarks in the design of primers. The PCR product (ArsR gene) must contain the start and stop codons if we wish to express it later.

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CHAPTER 6: Primer Design

reverse primer
ATG TAA

forward primer
Figure 6.1. Annealing of forward and reverse primers to template DNA. The start and stop codons are represented on the top sense strand. The forward primer does not necessarily have to anneal to the template DNA in the region of the start codon, but may be designed to anneal to template DNA upstream of the start codon. Such primer design may be done if these upstream DNA sequences are offered in the NCBI database. As the forward primer extends in PCR, the start codon is still incorporated in the PCR product. Why would a forward primer sometimes be designed to anneal to template DNA upstream of the start codon? If the region of template DNA around the start codon is too rich in As and Ts (more than 60%), a more efficient primer could be designed to anneal to template DNA somewhere upstream of the start codon, where the ratio of purines to pyrimidines is balanced. It is important, however, not to design a forward primer that anneals to template DNA downstream of the start codon, as the resulting truncated PCR product would not encode a functional protein. The same principle applies to the design of the reverse primer. As the reverse primer extends during PCR, it must incorporate the stop codon of the gene into the PCR product. If the ratio of purines to pyrimidines is more balanced downstream of the stop codon, then the reverse primer may be designed to anneal to that region of template DNA.

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CHAPTER 6: Primer Design

You have determined the general direction in which you want each primer to extend: forward primer from the start towards stop codon of the template DNA, and reverse primer from the stop towards the start codon. You also know that the primers should be complementary to the template DNA if PCR is to work. However, there are two strands of template DNA, sense and anti-sense. Which primer anneals to the sense strand of template DNA and which to the anit-sense strand of template DNA? Keep in mind that a designed primer is both complementary and antiparallel to the strand of template DNA it anneals to. Note that each primer has polarity: it begins with a 5’end, and during PCR is extended through the activity of Taq DNA polymerase by the addition of nucleotides at its 3’end. Each strand of template DNA also has a 5' and 3' end. Before continuing to read on, examine Figure 6.1 and decide whether the two primers are annealing properly to the strands of template DNA. As shown in Figure 6.1, the forward primer extends from the start to the stop codon if it is complementary and anneals to the anti-sense strand of the template DNA. This is because the 5' end of the forward primer can "face" only the 3' end of the anti-sense strand of template DNA (not the 5' end of the sense strand of template DNA!) as they anneal in an antiparallel fashion. The reverse primer is complementary and anneals to the sense strand of template DNA. The 5' end of the reverse primer can "face" only the 3' end of the sense strand of template DNA if the reverse primer is to extend from the stop towards the start codon of template DNA. Determining the Lengths of the Primers Primers are usually 20-30 nucleotides long and the length of the primers depends on the nucleotide content of the template. Primers should contain about 40-60% of Gs and Cs and the melting temperature (Tm) of a primer should fall in the range of 55-65 ºC. To satisfy the requirements of Tm, a primer with 60% As and Ts is usually designed to be longer than a primer with 40% As and Ts. The Tm of forward and reverse primers used in PCR should differ by no more than 2ºC. Refer to Chapter 5, page 37 on the calculation of the Tm of primers.

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HOT TIP!
The sequence of the forward primer corresponds to sense DNA! Introduction of Restriction Sites into Primers Following the basic rules of primer design, the primers that you have designed, will anneal to the K12 E. coli genomic DNA during PCR to amplify the desired ArsR gene. These PCR products will be introduced into plasmids for future manipulations. Plasmids are doublestranded circular DNA molecules found naturally in bacteria (see Chapter 2). However, plasmids used in research and the biotech industry have been genetically manipulated to contain several useful features: the MCS (multiple cloning site), several promoters and antibiotic-resistance genes, such as Amp . These plasmids are often referred to as vectors. Genes amplified in PCR are inserted into the MCS of a plasmid to create a recombinant plasmid. Recombinant plasmids are circular DNA molecules composed of DNA from different sources: in addition to the commercial plasmid DNA, recombinant plasmids contain the inserted gene, referred to as “foreign,” as it may originate from just about any species. Expression of the foreign gene is controlled through the activity of the plasmid promoter. The MCS of a plasmid provides a variety of restriction sites (recognition sequences). These are unique restriction sites, meaning that they are not repeated elsewhere in the plasmid sequence. A foreign gene is introduced into one, or more often, two of these unique restriction sites. Plasmids must be linearized, or cut by one or two restriction enzymes before a foreign gene may be introduced into the MCS. Some restriction enzymes such as Eco RV create blunt ends in cut DNA, while the majority of enzymes create overhanging or sticky ends (eg. Bam H1 and Eco R1) in cut DNA. See Figure 6.2.
R

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CHAPTER 6: Primer Design

EcoRV

BamH1

EcoR1

Figure 6.2. Restriction sites of EcoRV, BamH1, and EcoR1 restriction enzymes.

There are drawbacks in using only one restriction enzyme when attempting to introduce a foreign gene. During ligation (see Chapter 10), it is not possible to control the orientation of the gene that will be integrated into the plasmid. If the goal of introducing a gene into a plasmid is to express the gene, then orientation of the gene (and thus the positions of the start and stop codons) relative to the promoter of the plasmid is crucial. When inserted into a plasmid, the start codon of a gene should be located directly after the plasmid promoter. Proper orientation of a gene within the plasmid may be controlled by introducing a different restriction site at either end of the PCR product. Both the PCR product and the plasmid can be cut at these two restriction sites, thus allowing the joining (ligation) of the gene and the cut plasmid in the desired orientation. For example, we may decide to introduce the ArsR gene into the Sal 1 and Xba 1 restriction sites of a plasmid and then express the gene. In this recombinant plasmid, the start codon of the ArsR gene should be flanked by the Sal 1 restriction site of plasmid and the stop codon of the ArsR gene should be flanked by the Xba 1 restriction site of plasmid. This position of ArsR gene in the recombinant plasmid would bring the start codon of the ArsR gene in the proximity of the plasmid promoter (because Sal 1 is closer to the plasmid promoter than Xba 1) and ensure proper expression of the gene. To perform sticky-end ligation, both the plasmid and the PCR product should possess identical sticky ends after they have been cut with restriction enzymes. Based on this information, it is important to incorporate the same restriction sites into the primers as the ones being cut in plasmid. How we want our gene positioned with respect to the plasmid promoter determines which restriction site is incorporated into which primer during primer design. If the ArsR gene is to be introduced into the Sal 1 and Xba 1 restriction sites of a plasmid and placed under control of the plasmid promoter, then the forward primer should be designed to contain
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CHAPTER 6: Primer Design

the Sal 1 restriction site, while the reverse primer should be designed to contain the Xba 1 restriction site. Design of Forward Primer You may choose restriction sites in the MCS. Not every restriction enzyme in the MCS of pETBlue-2 is available in this lab, but there are enough choices where you can pick your own pair of restriction enzymes. When choosing which restriction sites you are going to use in the MCS, it is important to take a few things into consideration: 1. One or both of the restriction enzymes you choose in the MCS may also cut ArsR. Verify that ArsR does not contain restriction sites of either of your chosen restriction enzymes. 2. Each enzyme requires its own optimal reaction buffer. Some restriction enzymes can use the same buffer. It can save a bit of time if both of your enzymes can work on the DNA at the same time, but this is not required. It is straightforward to digest the DNA first with enzyme 1, clean up the DNA, and then digest the DNA with enzyme 2. 3. Make sure that the restriction sites you choose maintain ArsR in-frame with the start codon of pETBlue-2. If ArsR is not in-frame, then it will not be expressed as a functional protein. How are restriction sites added to each primer? Remember that the 3’ portion of the primer must be complementary to the template DNA. Therefore, the restriction site is introduced at the 5’ end of the primer. For example, you may have previously designed the forward primer for amplification of the ArsR gene as follows: forward primer: 5’ ATG GGA GTC AAA GTT CTG TTT G 3’ start codon

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The Sal 1 restriction site, 5’ GTC GAC 3’, is simply added upstream of the start codon in the forward primer:

forward primer with restriction site:

5’ GTC GAC ATG GGA GTC AAA GTT CTG TTT G 3’ Sal 1 restriction site

The orientation of the restriction site has to be consistent with the rest of the primer and must be written in the 5’ to 3’ direction. If the above forward primer was used in PCR, the resulting PCR product would be impossible to digest with Sal 1 and subsequent introduction of the PCR product into the plasmid would fail. The restriction enzyme requires additional structural “support” for binding and cutting of DNA, which must be provided by neighboring nucleotides. Thus, it is usually sufficient if any two to four nucleotides are added at the 5’ end of the primer, upstream of the restriction site:

complete forward primer: 5’ GCA GTC GAC ATG GGA GTC AAA GTT CTG TTT G 3’ Sal 1 restriction site

HOT TIP!
Restriction site nucleotide sequences may be found in catalogs or websites of biotech companies such as New England Biolabs, Fermentas, Invitrogen or Stratagene. Check them out!

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CHAPTER 6: Primer Design Design of Reverse Primer Adding a restriction site to the reverse primer is tricky. Please try this exercise before checking your result in Figure 6.5. 1. Using only lines, draw a rough schematic of both strands of the template DNA and the primers. Label the 5' and 3' ends of the template strands and the primers. Determine the direction in which the reverse primer extends. Is the reverse primer complementary to the sense or antisense template DNA strand? 2. Refer to the published DNA sequence of the ArsR gene (pages 58-59) and locate the stop codon. The ArsR gene is the template DNA. 3. Look at the DNA sequence around the stop codon of the ArsR gene. On a piece of paper, copy about 15 of these bases, which are on the sense DNA strand. Below this sequence, write the complementary sequence. Make sure the stop codon is included in your sequence. Label 5' and 3' ends of both DNA strands. The complementary sequence you wrote is your incomplete reverse primer. 4. Compare your result with Figure 6.5.

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CHAPTER 6: Primer Design sense strand 5’ ATG reverse primer cgg cca cca 3’ GCC GGT GGT cta att 5’ GAT TAA 3’ stop codon

anti-sense strand 3’ TAC

ATT 5’

Figure 6.5. Annealing of the reverse primer to the template DNA. The template DNA bases are represented by bold letters and bold horizontal lines. The reverse primer is shown in italics. So far you've designed a reverse primer that is only complementary to the template DNA, but has no restriction site on its 5' end. The following step is to write the reverse primer in the 5' to 3' direction. Always write your primers in this direction, and label the 5' and 3' ends. reverse primer : 5' TTA ATC ACC ACC CGG 3' Add the Xba 1 restriction site sequence (5' TCT AGA 3') to the 5' end of the primer, preserving the 5' to 3' orientation of both the restriction site and the primer: reverse primer with restriction site : 5' TCT AGA TTA ATC ACC ACC CGG 3' Xba 1 restriction site At the 5' end of primer, add at least two extra bases. complete reverse primer : 5' CAG TCT AGA TTA ATC ACC ACC CGG 3' Xba 1 restriction site

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CHAPTER 6: Primer Design Great! You have designed your first reverse primer! Have a careful look at the primer. Does it have any flaws? What about the GC content? Does the Tm compare to that of the forward primer? Important note: when calculating the Tms of primers, count only the values of the primer bases (2 for A and T, 4 for G and C) that are complementary to template DNA. Do not incorporate the restriction site into your calculation. To calculate the Tm of the reverse primer in the examples above, only consider the following bases of the reverse primer: 5' TTA TTT TGC GTC AGC 3'.

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CHAPTER 6: Primer Design
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Comment Features Sequence LOCUS NC_000913 354 bp DNA linear BCT 01-MAY-2007 DEFINITION Escherichia coli K12, complete genome. ACCESSION NC_000913 REGION: 3646551..3646904 VERSION NC_000913.2 GI:49175990 PROJECT GenomeProject:225 KEYWORDS . SOURCE Escherichia coli K12 ORGANISM Escherichia coli K12 Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Escherichia. Guy Plunkett III, guy@genome.wisc.edu), EcoGene (contact: Dr. Kenneth Rudd, ecogene@med.miami.edu) and the Coli Genetic Stock Center (contact: Dr. Mary Berlyn, mary.berlyn@yale.edu). COMPLETENESS: full length. FEATURES Location/Qualifiers source 1..354 /organism="Escherichia coli K12" /mol_type="genomic DNA" /strain="K-12" /sub_strain="MG1655" /db_xref="taxon:83333" gene 1..354 /gene="arsR" /locus_tag="b3501" /note="synonyms: ECK3486, arsE, JW3468" /db_xref="ECOCYC:EG12235" /db_xref="EcoGene:EG12235" /db_xref="GeneID:948013" CDS 1..354 /gene="arsR" /locus_tag="b3501" /function="regulator; Drug/analog sensitivity" /GO_component="cytoplasm" /GO_function="transcriptional repressor activity" /GO_process="response to drug; transcription" /note="transcriptional repressor of chromosomal ars operon" /codon_start=1 /transl_table=11 /product="DNA-binding transcriptional repressor" 58

CHAPTER 6: Primer Design
/protein_id="NP_417958.1" /db_xref="GI:16131373" /db_xref="ASAP:ABE-0011435" /db_xref="UniProtKB/Swiss-Prot:P37309" /db_xref="ECOCYC:EG12235" /db_xref="EcoGene:EG12235" /db_xref="GeneID:948013" /translation="MSFLLPIQLFKILADETRLGIVLLLSELGELCVCDLCTALDQSQ PKISRHLALLRESGLLLDRKQGKWVHYRLSPHIPAWAAKIIDEAWRCEQEKVQAIVRN LARQNCSGDSKNICS" ORIGIN 1 61 121 181 241 301 //
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atgtcatttc atcgttttac gaccagtcgc ctggaccgca gcggcgaaaa cgcaacctgg

tgttacccat tgctcagcga agcccaagat agcaaggtaa ttattgatga ctcgacaaaa

ccaattgttc actgggagag ctcccgccac gtgggttcat ggcctggcga ctgttccggg

aaaattcttg ttatgcgtct ctggcattgc taccgcttat tgtgaacagg gacagtaaga

ctgatgaaac gcgatctctg tgcgtgaaag caccgcatat aaaaggttca acatttgcag

ccgtctgggc cactgctctc cgggctattg tccagcatgg ggcgattgtc ttaa

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