p53 Testing for Li-Fraumeni and Li-Fraumeni-Like Syndromes
Kelly Gonzalez,1 Cindy Fong,1 Carolyn Buzin,1 Steve S. Sommer,1 and Juan-Sebastian Saldivar1
1City of Hope National Medical Center, Duarte, California
ABSTRACT
Li-Fraumeni Syndrome (LFS; OMIM #151623) is an autosomal dominant cancer pre- disposition syndrome characterized by early onset tumors including sarcomas, breast cancer, leukemia, brain tumors, and adrenocortical carcinoma. Li-Fraumeni syndrome is primarily attributed to germline mutations in the p53 tumor suppressor gene, which encodes tumor protein 53. In addition to germline p53 mutations, the p53 gene is the most commonly mutated gene in human cancers, with as much as 50% of tumors containing somatic p53 mutations. This unit provides a protocol to perform germline mutation anal- ysis of the p53 gene. The protocol includes steps for amplification and sequencing of the entire coding region of the p53 gene (exons 2 to 11). The protocol was designed for detecting germline alterations from DNA extracted from blood; however, with some ad- ditional optimization, it could also be used to detect somatic mutations in DNA extracted from tumors. Curr. Protoc. Hum. Genet. 57:10.10.1-10.10.11. @ 2008 by John Wiley & Sons, Inc.
Keywords: p53 . TP53 · Li-Fraumeni Syndrome
INTRODUCTION
This unit describes a technique to perform sequencing analysis of the p53 gene. Detection of a mutation in a patient with Li-Fraumeni or Li-Fraumeni-like syndrome confirms the clinical diagnosis and provides a useful tool to screen at-risk family members. This protocol is for individuals who are interested in sequencing the p53 gene to detect germline mutations associated with Li-Fraumeni and Li-Fraumeni-like syndromes.
The process begins with PCR amplification and purification of the coding regions of the p53 gene (Basic Protocol 1). These amplicons are then sequenced and analyzed for mutations (Basic Protocols 2 and 3).
These techniques can be applied to the sequencing analysis of other genes. However, de- sign of the amplification and sequencing primers, and optimization of cycling parameters, will have to be determined individually for each gene of interest.
PCR AMPLIFICATION OF EXONS 2 TO 11 OF THE p53 GENE AND PCR PRODUCT PURIFICATION
The p53 gene, located at 17p13.1, is composed of 11 exons, of which only exons 2 to 11 are coding. Mutations in the coding region and associated splice sites are associated with Li-Fraumeni and Li-Fraumeni-like syndromes. This protocol describes how to amplify the coding exons and their associated splice site junctions. Taking advantage of some short intronic sequences, exons 2 to 4, 5 to 6, 7, 8 to 9, and 10 to 11 are amplified as single amplicons (Fig. 10.10.1). The optimization for this approach requires the use of different master mixes, but only a single thermal cycling program. Once the appropriate exons have been successfully amplified and confirmed by agarose gel electrophoresis, purification of the product is performed to remove any remaining PCR amplification reagents. This
BASIC PROTOCOL 1
p53 gene
exon 1
exon 11
Cen
Tel
0
12 kb
20 kb
helps ensure that the cycle sequencing reaction occurs efficiently and without inhibition. A number of methods can be used for this process, including commercially available kits. This protocol describes how to purify the PCR products using an exonuclease I and shrimp alkaline phosphatase digestion protocol. Exonuclease removes all single- stranded materials such as primers and extraneous single-stranded products. Shrimp alkaline phosphatase removes any excess dNTPs. The double-stranded PCR product is preserved.
Materials
10× PCR buffer with 15 mM MgCl2 (comes with AmpliTaq enzyme from Roche)
1.25 mM dNTP mix (1.25 mM each dNTP)
2.5 µM PCR primer mixes (see Table 10.10.1): mixes of upstream and downstream primers for each amplicon, with each primer at 2.5 uM
5 U/ul AmpliTaq Gold polymerase (Roche)
PCR-grade H2O
50% DMSO
25 mM MgCl2
50 ng/ml genomic DNA (APPENDIX 3B)
Sterile water
1 × TAE buffer (APPENDIX 2D)
10 U/ul exonuclease I enzyme (Exo; from USB)
2 U/ul shrimp alkaline phosphatase enzyme (SAP; from USB)
200 ul PCR tubes
Thermal cycler (Applied Biosystems 9700 or equivalent)
Additional reagents and equipment for agarose gel electrophoresis (UNIT 2.7)
Perform PCR amplification
1. Prepare the appropriate amount of master mix (total 24.0 ul per reaction) by com- bining the following volumes:
Exons 2 to 4 and 10 to 11:
2.5 ul 10× PCR buffer (1x final concentration)
4.0 ul 1.25 mM dNTP mix (200 µM final concentration)
1.0 ul 2.5 uM primer mix (0.1 uM final concentration)
0.2 ul Taq Gold
16.3 ul PCR-grade water.
Exons 5 to 6:
5.0 ul DMSO (10% final concentration)
1.0 ul 25 mM MgCl2 (1 mM final concentration)
2.5 ul 10× PCR buffer (1 x final concentration)
4.0 ul 1.25 mM dNTP mix (200 uM final concentration)
4.0 ul primer mix (0.4 uM final concentration)
0.2 ul Taq Gold
7.3 ul of PCR-grade water.
p53 Testing for Li-Fraumeni and Li-Fraumeni-Like Syndromes
10.10.2
| Exon | Primer | Primer sequence 5'->3' | Primer length | Fragment size |
|---|---|---|---|---|
| 2-4 | P53E2-4PD | GGG TTG TGG TGA AAC ATT GGA AGA | 24 | 1030 bp |
| P53E2-4PU | GCT GAG GGT GTG ATG GGA TGG | 21 | ||
| 5-6 | P53E5-6PD | GCT CCT GAG GTG TAG ACG CCA A | 22 | 586 bp |
| P53E5-6PU | GCC ACT GAC AAC CAC CCT TAA C | 22 | ||
| 7 | P53E7PD | CAA GGC GCA CTG GCC TCA | 18 | 194 bp |
| P53E7PU | GGC ACA GCA GGC CAG TGT | 18 | ||
| 8-9 | P53E8-9PD | TGG GẠC AGG TAG GAC CTG AT | 20 | 505 bp |
| P53E8-9PU | CCA GGA GCC ATT GTC TTT GAG GCA TCA CT | 29 | ||
| 10-11 | P53E10-11PD | GCT GTA TAG GTA CTT GAA GTG CAG TTT CT | 29 | 1416 bp |
| P53E10-11PU | AGG CCA ACT TGT TCA GTG GAG C | 22 |
Exons 7 and 8 to 9:
5.0 ul 50% (v/v) DMSO (10% final concentration)
1.0 ul 25 mM MgCl2 (1 mM final concentration)
2.5 ul 10× PCR buffer (1x final concentration)
4.0 ul 1.25 mM dNTP mix (200 uM final concentration)
2.0 ul primer mix (0.2 uM final concentration)
0.2 ul Taq Gold
9.3 ul of PCR-grade water.
Vortex the master mix gently and spin down quickly using a benchtop centrifuge for 5 sec. Store on ice.
See Table 10.10.1 for detailed primer information.
2. Aliquot 24.0 ul master mix into a 200-ul PCR tube for each amplicon (one tube per amplicon per sample, and an additional tube per amplicon for negative controls).
Steps 1 through 3 should be done in a clean room to avoid contamination.
3. For each sample, add 1 ul genomic DNA directly into the master mixture in the 200-ul PCR tubes for each amplicon to be amplified.
4. For controls, add 1 ul sterile water for each amplicon to be amplified. These will serve as controls for contamination.
5. Place the tubes into a thermal cycler and amplify as follows:
| 1 cycle: | 10 min | 95°℃ | (denaturation) |
| 35 cycles: | 30 sec | 95°℃ | (denaturation) |
| 30 sec | 58℃ | (annealing) | |
| 60 sec | 72°C | (extension) | |
| 1 cycle: | 10 min | 72°C | (extension) |
| Final step: | indefinitely | 4°℃ | (hold). |
Optimization may be required as thermal cycler temperatures vary. This protocol has been optimized on an ABI 9700 96-well thermal cycler.
6. Check for amplification by performing electrophoresis on 4 ul of each reaction on a 2% agarose gel in 1 x TAE buffer, using a DNA ladder marker (UNIT 2.7).
7. Check samples for amplification and verify that the negative control lanes (those containing sterile water) did not show any evidence of amplification.
If a band is present in the control lanes, this indicates that contamination has occurred and the PCR amplification must be repeated. In this case, the use of new working solutions for all reagents is recommended in order to minimize the possibility of repeat contamination.
If no amplification is seen in the reactions with DNA, or if only a faint band is detected, this indicates poor amplification and the PCR amplification can be repeated as previously described.
Purify PCR products
8. For each PCR product, place 5 ul reaction mix in a new 200-ul tube and add 1 ul of 10 U/ul exonuclease I and 1 ul of 2 U/ul shrimp alkaline phosphatase. Pipet 2 to 3 times to mix.
9. Place the tubes into the thermal cycler and treat as follows:
| 15 min | 37°℃ |
| 15 min | 80°℃ |
| hold | 4°C. |
Water baths with the described temperatures can also be used.
PCR products can be stored at -20℃ for an extended period of time (1 year or possibly longer).
BASIC PROTOCOL 2
CYCLE SEQUENCING OF PURIFIED PCR PRODUCTS
Direct sequencing of genetic material is the current gold standard for detecting point mutations and small deletions, duplications, or insertions. This protocol provides thermal cycling parameters for fluorescent cycle sequencing of the purified PCR products. Again, taking advantage of some short intronic sequences, exons 2 to 3, 4, 5 to 6, 7, 8 to 9, 10, and 11 are sequenced in individual cycle sequencing reactions. There are several companies that sell cycle sequencing kits. This protocol has been developed using ABI’s BigDye Terminator Kit v1.1 on an ABI 9700 thermal cycler.
As with PCR amplification, cycle sequencing reactions are left with some residual reagents that must be removed in order to ensure the sequencing data are rich and clean. There are a number of methods for cycle sequencing purification, from homemade column purification techniques to commercially available vacuum, column, and magnetic bead purification kits. This protocol presents the SeqGen magnetic bead purification method from Agencourt.
Materials
ABI BigDye Terminator Kit v1.1 and 5x sequencing buffer (Applied Biosystems) Milli-Q H2O
Purified PCR products (see Basic Protocol 1)
1.5 mM sequencing primers (see Table 10.10.2)
CleanSeq kit (Agencourt) including: Magnetic bead solution Magnetic purification beads
85% ethanol (freshly made)
0.3 mM EDTA (elution buffer)
Thermal cycler (Applied Biosystems 9700 or equivalent) Magnetic purification tray (Agencourt) 200-ul pipet 10-ul tip
p53 Testing for Li-Fraumeni and Li-Fraumeni-Like Syndromes
10.10.4
| Exon | Primer | Primer sequence 5'->3' | Primer length | |
|---|---|---|---|---|
| 2-3 | P53E2-3SD | TGT CTC AGA CAC TGG CAT GG | 20 nt | |
| P53E2-3SU | TGA AAA GAG CAG TCA GAG GAC | 21 nt | ||
| 4 | P53E4SD | AAG GGT TGG GCT GGG GAC CT | 20 nt | |
| P53E4SU | AGT TCC AAA CAA AAG AAA TGC AG | 23 nt | ||
| 5-6 | P53E5-6SD | GCT CCT GAG GTG TAG ACG CCA A | 22 nt | |
| P53E5-6SU | ACT TTG CAC ATC TCA TGG GG | 20 nt | ||
| 7 | P53E7SD | CAA GGC GCA CTG GCC TCA | 18 nt | |
| P53E7SU | GGC ACA GCA GGC CAG TGT | 18 nt | ||
| 8-9 | P53E8-9SD | TGG GẠC AGG TAG GAC CTG AT | 20 nt | |
| P53E8-9SU | CCA GGA GCC ATT GTC TTT GAG GCA TCA CT | 29 nt | ||
| 10 | P53E10SD | CAA TTG TAA CTT GAA CCA TCT TT | 23 nt | |
| P53E10SU | GGC AGG ATG AGA ATG GAA TC | 20 nt | ||
| 11 | P53E11SD | GCA CAG ACC CTC TCA CTC A | 19 nt | |
| P53E11SU | CAA AAT GGC AGG GGA GGG A | 19 nt | ||
Perform cycle sequencing
1. Prepare the appropriate amount of master sequencing mix (total 5.9 ul per reaction):
2.0 ul 5× buffer (1x final concentration)
0.4 ul BigDye Terminator v1.1 (4% final concentration)
3.5 ul of Milli-Q H2O.
Place on ice.
Each exon to be sequenced requires two sequencing reactions, one for the sense strand and one for the antisense strand.
2. Label two PCR tubes per exon per sample (one for each direction of sequencing).
3. In each tube, put:
3.1 ul purified PCR product
1.0 ul sequencing primer (0.15 mM final concentration)
See Table 10.10.2 for detailed primer information.
4. Aliquot 5.9 ul sequencing mix into each tube (total reaction volume 10 ul).
5. Vortex quickly and spin down the tubes on a benchtop centrifuge for ~5 sec.
6. Cycle sequence on a thermal cycler using the following conditions:
| 1 cycle: | 1 min | 94℃ | (denaturation) |
| 35 cycles: | 15 sec | 94℃ | (denaturation) |
| 30 sec | 55°℃ | (annealing) | |
| 60 sec | 72°C | (extension) | |
| 1 cycle: | 10 min | 72°C | (extension) |
| Final step: | indefinitely | 4ºC | (hold). |
All samples should be sequenced and analyzed in both the sense and antisense directions.
Optimization may be required as thermal cycler temperatures vary. This protocol has been optimized on an ABI 9700 96-well thermal cycler.
Purify sequencing reactions
7. Briefly microcentrifuge cycle-sequenced samples.
8. Gently shake the bottle of CleanSeq magnetic bead solution to resuspend the mag- netic particles.
9. Add 10 ul magnetic bead solution to each cycle-sequenced reaction tube.
10. Add 42 ul of 85% ethanol to each reaction tube and mix 5 times. Let stand for 30 sec at room temperature.
11. Place the tubes onto the magnetic purification tray and let stand for 5 min at room temperature.
12. Aspirate as much of the clear supernatant solution as possible from each tube using a 200-ul pipet and discard.
13. Repeat aspiration, using a 10-ul tip to remove any residual supernatant from the tubes.
14. Dispense 100 ul of 85% ethanol into each tube and incubate for 30 sec at room temperature.
15. Aspirate supernatant as in steps 12 and 13.
16. Remove the tube from the magnetic purification tray and allow the tubes to stand at room temperature for 10 min to evaporate any residual ethanol.
17. Add 40 ul of 0.3 mM EDTA to the tubes and let stand for 5 min at room temperature.
18. Place tubes back on the magnetic purification tray and let stand for 5 min at room temperature.
19. Remove 30 ul of the eluted sample and dispense into a clean tube or tray.
20. Store samples in a dark environment at 4℃ if not running samples immediately. Samples should ideally be analyzed within 48 to 72 hr due to degradation of the fluorescent signal.
BASIC PROTOCOL 3
SEQUENCE ANALYSIS
Automated fluorescent sequencing has significantly improved what was once a very time-consuming process that required the use of radioactive materials and the running of large slab gels. Presently, there are a number of automated sequencing platforms that can expedite the sequencing process, and a variety of programs that allow direct comparison of the sequencing data to established wild-type sequence. This protocol was developed using an ABI 3730 automated capillary sequencer and Sequencher v4.2 analysis software.
Materials
Purified cycle sequence products (see Basic Protocol 2) Automated sequencer (ABI 3730 from Applied Biosystems or equivalent) Sequence analysis software (Sequencher by Gene Codes or equivalent)
1. Run the purified sequencing products on the ABI 3730 automated sequencer accord- ing to manufacturer’s instructions.
2. Input the known wild-type sequence for the fragments being analyzed into the Sequencher program.
p53 Testing for Li-Fraumeni and Li-Fraumeni-Like Syndromes
10.10.6
3. Import the sequencing data for the samples and compare directly to the established wild-type control sequence.
The program will flag any discrepancies it detects. However, caution should be used to avoid relying too heavily on this flag, as these programs are by no means 100% accurate.
4. Perform a direct visual inspection of the sequencing chromatograms, looking for subtle heterozygous peaks that the program may have missed.
All exons should be sequenced in both the sense and antisense directions. If robust amplification occurs, mutations should be detected in both directions, confirming a true mutation.
Some academic centers have a sequencing core facility where the cycle sequencing and analysis protocols can be outsourced. Contact the core facility for specific sample submission requirements.
COMMENTARY
Background Information
Li-Fraumeni Syndrome (LFS; OMIM #151623) is an autosomal dominant can- cer predisposition syndrome characterized by early onset tumors. The five main cancers as- sociated with LFS are sarcoma, breast can- cer, leukemia, brain tumors, and adrenocorti- cal carcinoma (Garber et al., 1991); however, many other types of tumors are associated with Li-Fraumeni syndrome (Varley, 2003). The lifetime penetrance of LFS is high (Nagy et al., 2004). Because of the predominance of breast cancer associated with LFS, females have an overall higher risk of developing cancer and an earlier age of onset. The risk for cancer by age 50 is 93% in women and 68% in men, and the average age of onset is 29 years in women and 40 years in men (Hwang et al., 2003).
Li-Fraumeni syndrome is primarily at- tributed to germline mutations in the p53 tu- mor suppressor gene, which encodes tumor protein 53 (TP53; Malkin et al., 1990). Un- like some cancer predisposition syndromes in which the majority of the mutations cause protein truncation, the majority of the muta- tions in LFS are missense mutations (Varley, 2003). The p53 gene has been thought to me- diate the balance between mutagenesis, repair, and apoptosis, and has been proposed as the “guardian of the genome” (Lane, 1992). How- ever, it has been reported that mutation fre- quency and pattern in Big Blue p53-/- mice are essentially the same as those in Big Blue p53+/+ mice, with the exception of chronoco- ordinate doublet mutations and perhaps mu- tation showers (Hill et al., 2006; Wang et al., 2007). This gene encodes a transcription fac- tor important in the regulation of cell prolifer- ation, implicating its role in tumorigenesis. In response to DNA damage, TP53 is involved in cell cycle checkpoint regulation (Shiloh,
2001). Depending on the severity of the DNA damage, TP53 has been shown to play a role in either the induction of DNA repair (Ford et al., 1995), cellular senescence (Han et al., 2002), or apoptosis (Yonish-Rouach et al., 1991; Kaufmann and Earnshaw, 2000). Mice defi- cient in TP53 show increased levels of DNA adducts in response to mutagens (Carmichael et al., 2001) and increased levels of chromoso- mal instability (Li et al., 1988). While the role of TP53 in tumorigenesis is clear, the function is still not completely understood (reviewed in Morris, 2002).
Mutations in a second gene, CHEK2, oc- cur in a minority of patients with Li-Fraumeni syndrome (Bell et al., 1999). CHEK2 is a cell cycle protein that responds to DNA damage by phosphorylating p53 and BRCA1 (Bartek et al., 2001).
What Li and colleagues first described based on the analysis of the first 24 reported families (Li et al., 1998) would later be known as classic LFS (Garber et al., 1991). Classic LFS has been associated with a very high p53 mutation detection rate, with ~70% to 80% of families meeting classic LFS crite- ria having a p53 mutation (Eng et al., 2001; Strong, 2003; Varley, 2003; Nagy et al., 2004; Libe and Bertherat, 2005). The more inclu- sive Birch and Eeles clinical criteria were later defined and are collectively described as Li- Fraumeni-like syndrome (LFL; Birch et al., 1994; Eeles, 1995). p53 mutation detection rates for LFL range from 7% to 40% (Birch et al., 1994; Eeles, 1995; Varley et al., 1997; Varley, 2003). Specifically, the Birch classi- fication shows slightly higher detection rates than Eeles, ranging from 11% to 40% and 7% to 8%, respectively. The Chompret classifica- tion, developed in 2001, uses an extensive al- gorithm for prediction of p53 mutation status
10.10.7
(Chompret et al., 2000; Chompret et al., 2001). Of families meeting Chompret classification, 20% had p53 mutations, and the classifica- tion was reported to have a sensitivity of 80% (Chompret et al., 2001).
The spectrum of cancers occurring in Li- Fraumeni syndrome extends beyond the orig- inally described “core” cancers. Several case reports show that choroid plexus carcinomas (CPC) are associated with LFS (Garber et al., 1990; Sedlacek et al., 1998). Krutilkova sug- gests that patients with CPC should be re- ferred for p53 testing regardless of family his- tory based on the identification of five patients with CPC and p53 mutations (Krutilkova et al., 2005). Similarly, an analysis of the p53 gene in patients ascertained because of a diagnosis of childhood ACC yielded an 82% mutation de- tection rate (Varley et al., 1999). An analysis of the prevalence of early onset colon cancer in patients with classic LFS reveals that 2.8% (11/397) of patients with classic LFS had colon cancer prior to age 50 with a mean age of diag- nosis of 33 years (Wong et al., 2006). Based on a review of the spectrum of cancers in a cohort of 28 LFS families, it is noted that adrenocor- tical carcinoma, phyllodes tumor, and Wilm’s tumor, in addition to breast cancer, brain can- cer, and sarcoma, are also strongly associ- ated (Birch et al., 2001). Pancreatic cancer is moderately associated and leukemia may be weakly associated, despite the original obser- vation by Li et al. in their description of classic LFS (Garber et al., 1991).
The p53 gene is the most commonly mu- tated gene in human cancers, with up to 50% of tumors containing p53 somatic mutations (re- viewed in Hussain and Harris, 2006). A similar mutation pattern is observed in both somatic and germline p53 mutations, with the majority of mutations being missense and occurring in the core DNA binding domain (exons 5 to 8; Frebourg et al., 1995; Hainaut and Hollstein, 2000). Five “hotspot codons” (175, 245, 248, 273, and 282) are observed in both somatic and germline p53 mutations (Petitjean et al., 2007).
While a standardized recommendation for cancer screening for patients with p53 germline mutations remains lacking, the util- ity of genetic testing for both affected and presymptomatic patients is now a generally accepted practice. Symptomatic LFS patients have a significant risk for subsequent malig- nancies, reaching as high as a 57% lifetime risk for a second primary (Hisada et al., 1998). Well-established screening recommendations exist for breast cancer, the most common ma-
lignancy in LFS. Breast cancer screening for patients with p53 mutations begins at the age of 25 and includes clinical breast monitoring and annual breast MRI (Moule et al., 2006; Field et al., 2007). Additionally, prophylactic mastectomy can be offered to presymptomatic p53 mutation carriers (Moule et al., 2006). It has been suggested that children with p53 germline mutations should undergo annual physical exams (Field et al., 2007). Perhaps the most compelling reason to offer p53 test- ing for symptomatic patients is to aid in cancer treatment. It is generally recommended that patients with germline p53 mutations avoid radiotherapy due to a significant risk for a secondary malignancy in the irradiated area (Varley et al., 1999; Limacher et al., 2001; Moule et al., 2006; Field et al., 2007; Salmon et al., 2007).
Critical Parameters and Troubleshooting
The biggest issue with any PCR-based method is contamination, which can give false results. This is monitored by always including a negative control that contains all reagents for the amplification reaction, except that the DNA is replaced by water. Maintaining sepa- rate pre- and post-PCR working areas can help minimize contamination.
If DNA is extracted from blood, it is strongly recommended that the extraction be performed within 3 to 4 days of blood draw. Old or lysed blood samples will yield poor- quality DNA, which can prevent robust ampli- fication.
For additional troubleshooting tips, see Table 10.10.3.
Anticipated Results
The final output of this protocol is sequenc- ing data that cover the entire coding region and associated splice site junctions for exons 2 to 11 of the p53 gene (Fig. 10.10.1). Typ- ical results are either wild-type sequence, a benign polymorphism, or a mutation, which can include any deviation from the wild-type sequence. Some examples are a single nu- cleotide change or a small deletion, insertion, or duplication. It should be noted that cycle sequencing is unable to detect whole exon or multi-exon deletions, duplications, or inver- sions. While the large majority of reported p53 mutations are detectable by cycle sequencing, large deletions have also been described in a few cases. Detection of a large gene deletion requires an alternate method, such as compara- tive genomic hybridization (CGH; UNIT 4.6) or
p53 Testing for Li-Fraumeni and Li-Fraumeni-Like Syndromes
10.10.8
| Problem | Possible cause | Solution |
|---|---|---|
| Little or no amplification for entire assay | Poor DNA quality (old blood sample, hemolyzed sample, etc.) | Obtain fresh blood sample |
| DNA extraction technical error | Analyze DNA by agarose electrophoresis to determine if it is high molecular weight or degraded | |
| PCR/sequencing technical error | Reamplify/resequence once DNA quality is confirmed | |
| Poor amplification for a single amplicon | Rarely, an individual may have a polymorphism under one of the primers which may hinder amplification | Design new primers for that amplicon and repeat test |
| Uninterpretable sequencing data in exon 4 downstream and exons 2-3 upstream. Sequence appears as two different sequences overlying one another. | Occasionally, an individual may have a 16-bp insertion polymorphism in IVS3+40. This disrupts the reading frame on one of the alleles. | (1) Sequence exon 4 twice in the upstream direction and exons 2-3 twice in the downstream direction, or (2) design additional sequencing primers to avoid sequencing through the insertion. |
robust dosage PCR (RD-PCR; Liu et al., 2003; Nguyen et al., 2007). Previously reported al- terations in the p53 gene can be found on the IARC mutation database at http://www- p53.iarc.fr/. Two separate tables are main- tained at this site, one for germline mutations and the other for somatic mutations.
Sequence data should be of sufficient qual- ity and adequately free from background sig- nal for reliable results. Use of the Phred score is one way of objectively measuring quality.
Time Considerations
Each thermal cycling protocol takes ~3.5 hr, for both PCR and cycle sequencing. Analysis of the samples on an automated flu- orescent sequencer takes 1 to 2 hr, depending on the system being used. If a sequencing core is available for outsourcing this component, a 1- to 3-day turnaround time can be expected (check with the facility). Final analysis of the sequencing data can be done in an hour or less.
Acknowledgment
The authors would like to thank Jennifer Han for reviewing and updating the protocol details.
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p53 Testing for Li-Fraumeni and Li-Fraumeni-Like Syndromes
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Internet Resources
Information on p53 at the IARC mutation database. http://www.genecodes.com
Seqeuncher software, demo product available for download.