Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Genome Walking with 2- to 4-kb Steps Using Panhandle PCR Douglas H. Jones and Stanley C. Winistorfer Department of Pediatrics, University of Iowa College of Medicine, Iowa City, Iowa 52242-1083 Panhandle PCR is a method that per- The PCR permits the highly specific trastrand annealing, those single strands mits the rapid retrieval of DNA seg- amplification of DNA sequences, but re- of genomic DNA that contain the com- ments flanking a known DNA se- quires knowledge of the sequences that plement to the 3' ends wil! form a stem- quence. This method extends the flank both ends of the sequence of inter- loop structure with a recessed 3' end. application of PCR to the retrieval of est. ~1-3~ Several methods have been de- Therefore, the ligated oligonucleotide DNA where only one end of the DNA veloped to permit the PCR amplification can prime template-directed DNA poly- sequence is known, so that one can of unknown DNA that flanks one end of merization on those strands that contain use PCR to walk along an uncharac- a known sequence. (4-23) The number the known sequence, resulting in known terized stretch of DNA. In this report, and variety of methods developed to am- DNA being attached to the uncharacter- we demonstrate that this method can plify DNA flanking a known sequence at- ized end of the unknown DNA (Fig. 1, be applied to the highly specific re- test to the scientific need for an effective step 3). Because this polymerization re- trieval of large pieces (2.2-4.4 kb) of method. None of the existing methods, action results in known DNA being po- human genomic DNA that flank the apart from panhandle PCR, (23) has per- sitioned on both sides of the unknown primer annealing sites. mitted the highly specific PCR amplifi- flanking DNA, PCR can be used to am- cation of >2 kb of human genomic DNA plify the unknown DNA. that flanks a known site. This paper in- High specificity and yield are ob- troduces a protocol of panhandle PCR tained by use of a hot-start strategy prior that permits the highly specific PCR am- to generation of the initial PCR template plification of up to 4.4 kb of DNA flank- (shaped like a pan with a handle; Fig. 1, ing a known site directly from bulk hu- step 3), followed by a hot-start PCR am- man genomic DNA. The sequences plification, and then a nested hot-start retrieved were human genomic ~-globin PCR amplification. The rationale for sequence (24~ and the genomic DNA 5' to nested PCR amplification and for a hot- the human cystic fibrosis transmem- start strategy is detailed in two previous brane conductance regulator (CFTR) publications. ~1'28~ A hot start was accom- cDNA.(25-27) plished by not allowing the temperature The panhandle PCR method is illus- of the fully constituted polymerase reac- trated in Figure 1. The steps in Figure 1 tants to drop below 80°C before the an- are explained in detail in the Materials nealing step for priming, this tempera- and Methods section. Human genomic ture being well above the T m of any of DNA is digested with a restriction en- the primers. The hot start eliminates zyme that leaves a 5' overhang. This di- priming at low stringency, preventing gested DNA is treated with calf intestinal the generation of nonspecific products. alkaline phosphatase and the ends are li- The T m of the protruding single- gated to a 5' phosphorylated single- stranded 3' ends created by the ligated stranded oligonucleotide, so that the 3' oligonucleotide was calculated, and the end of each strand of digested genomic annealing temperature for forming the DNA is modified. The protruding single- stem-loop structure was set at 2°C below stranded 3' ends are complementary to a this T m value. The T m of each PCR region of known DNA that is upstream primer was also calculated, and the an- to the unknown region of interest. nealing temperature used in each PCR Therefore, when this complex mixture amplification was approximately 2°C be- undergoes denaturation and annealing low the mean T m of each primer set. Two under dilute conditions that promote in- primers are used in each PCR amplifica- 2:197-203©1993 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/93 $3.00 PCR Methods and Applications 197 Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press ;: 1~3 k ~ 2~ 4w ~ ..................... ,.,;:] primer 2 and 4). Use of one primer in Restriction Enzyme Digestion each amplification (primer 1 alone in Followed by Calf Intestinal Alkaline 1. Restriction enzyme digest the first PCR amplification and primer 3 Phosphatase Treatment r 5' in the second primer amplification) am- plified poorly (not shown), presumably 2. Ligate to -¢~At due to large inverted repeats in the ends (Step I) of each PCR product, which may com- 5" Five micrograms of human genomic pete with primer annealing. Primers 2 3. Add to Taq polymerase with dNTPs DNA (Clontech, Palo Alto, CA) was di- and 4 have 1 or 2 nucleotides added to gested with 20-40 units of a restriction the 5' end that are not homologous to enzyme that yields a 5' overhang (XbaI, the original template. This is done as a AvrII, BamHI, BclI, BglII, BspDI, BstBI, or precaution, to prevent short-circuiting re s' 3 5' 3, MluI; New England BioLabs, Beverly, of the amplification reaction. Such MA) in 100 i~l for 2 hr. The digested ge- short-circuiting could occur by anneal- nomic DNA was dephosphorylated by Intra-strand an merase ext ing of the 3' end of a strand of a short, the addition of 0.05 units of calf intesti- nonspecific PCR product (into which nal alkaline phosphatase in 5 i~l (Boe- primer 2 or 4 was incorporated) to the hringer Mannheim Biochemicals, India- targeted sequence, resulting in a short, napolis, IN) with incubation at 37°C for nonspecific product. Because this pre- 30 min. The DNA underwent glass bead caution is simple and inexpensive, its ne- extraction using Geneclean (BIO 101, La cessity has not been tested. This paper Jolla, CA), and was suspended in 50 ILl of 3' /dd primers illustrates that this method can amplify TE buffer (10 mM Tris-HC1, pH. 8, 1 mM l .¢, 2t.,¢ longer products, with higher specificity, EDTA). Five 5-1~I aliquots were frozen for 5, ~A r L than previously reported methods. later use as nonoligonucleotide-ligated template controls. $. PCR amplify • : t ............ .~ MATERIALS AND METHODS 5' Ligation of Phosphorylated 6. Transfer an aliquot to a Oligonucleotide Synthesis Oligonucleotide PCR mixture containing nested primers %,¢ Oligonucleotides were synthesized by Midland Certified Reagent Company (Step 2) (Midland, TX). Oligonucleotides with a ~ 7. PCR amplify phosphorylated 5' end were generated The remaining 25 ~l of genomic DNA was ligated to a 50-fold molar excess r 1 during oligonucleotide synthesis by ad- 5" dition of a phosphorylated phosphora- of a 5' phosphorylated oligonucleotide FIGURE 1 Panhandle PCR. The numbered midite. The sequences of the oligonucle- (whose 5' end is complementary to the steps correspond to the numbered steps in otides and Tm values are given in Tables single-stranded ends of restriction en- Materials and Methods. The two complemen- 1 and 2. Tm values were obtained using zyme-digested genomic DNA) in T 4 DNA tary strands of genomic DNA are thin and the computer program OLIGO 3,4 (Na- ligase buffer (50 mM Tris-HCl, pH 7.6, 10 thick lines. DNA that flanks the known region tional BioSciences, Hamel, NM). mM MgClz, 0.5 mM ATP, 10 mM dithio- of genomic DNA is enclosed by brackets and is striped when double-stranded. The jagged portion of the thick line represents the an- nealing region for the ligated oligonucle- otide. The PCR primers are numbered arrows. TABLE 1 Sequence of Oligonucleotides Used in Retrieval of Human Genomic The locations of the primers in relation to the [3-globin DNA relevant strands of genomic DNA are shown at top for step 1, and the primers are not used Oligonucleotide phosphorylated at the 5' end, ligated to XbaI-digested genomic DNA until step 4. One or two nucleotides are added 5'p-CTAGAGTCTTCTCTGTCTCCACATGCCCAGTTTCTATTGGTC. Tm of the 40-nucleotide- to the 5' ends of primers 2 and 4 that are not long 3' region of this oligonucleotide that is complementary to the known region of complementary to their template, and are genomic DNA is 71.5°C. represented by upended 5' ends. PCR amplification oligonucleotides Primer 1: GATGAAGTTGGTGGTGAGGCCCTG. T,~ 62.5°C Primer 2: GACTTGGGTTTCTGATAGGCACTGACTCTC. 5' tion: one that is homologous to the re- nucleotides that are not complementary to ~-globin gion upstream to the annealing site for are underlined; Tm 62.6°C; Tm minus 5' GA, 60.7°C the ligated oligonucleotide (Fig. 1, Primer 3 (nested primer): GTTGGTATCAAGGTTACAAGACAGGTTTAAGGAG.T m, primer 1 and 3) and one that is homol- 62.4°C. Primer 4 (nested primer): GACTGCCTATTGGTCTATITFCCCACCCTTAG. 5' ogous to a region located between the nucleotides not complementary to [3-globin are ligated oligonucleotide annealing site underlined; Tm, 64.5°C; Tm minus 5' GA, 62.9°C. and the unknown flanking DNA (Fig. 1, 198 PCR Methods and Applications Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press TABLE 2 Oligonucleotides Used to Retrieve Human Genomic DNA Flanking 5' End Set Up of Nested PCR Amplification of the CFTR cDNA Phosphorylated oligonucleotide ligated to Xba I or Avr II digested genomic DNA (Step 6) 5'p-CTAGCTTGAGCCCAGACGGCCCTAGCAGGGAC.