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Detecting Mutations in the APC Gene in Familial Adenomatous Polyposis (FAP)

Madhuri R. Hegde¹, Benjamin B. Roa¹

¹Baylor College of Medicine, Houston, Texas

Publication Name:

Current Protocols in Human Genetics

Unit Number:

Unit 10.8

DOI:

10.1002/0471142905.hg1008s50

Online Posting Date:

August, 2006

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Abstract

Hereditary forms of colorectal cancer (CRC) account for up to 5% of total cases. Familial adenomatous polyposis (FAP) is an autosomal dominant condition affecting nearly 1 in 5000 people and accounts for only about 1% of all CRCs. It is characterized by the progressive development of hundreds to thousands of adenomatous colon polyps. The gene associated with FAP (APC) contains 15 exons in the coding region. A scanning approach for large genes is reasonable, but some standard techniques have limited analytical sensitivity. The method described here, using DHPLC as mutation scanning approach for medium-throughput DNA sequence analysis, is largely considered to be the gold standard for point mutation analysis, and can be optimized for high-throughput testing. Detection of deletion and duplication mutations refractory to sequencing have been described using real-time quantitative PCR for dosage analysis. Technical strategies for mutation detection in the APC gene are presented in this chapter.

Keywords: APC gene; mutation detection; mutation scanning; DHPLC; Sequencing

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Unit Introduction
Basic Protocol 1: APC Gene Mutation Analysis by DHPLC and Direct Sequencing
Support Protocol 1: Preparation of Sample DNA from Whole Blood
Support Protocol 2: Quantitation of Genomic DNA by Spectrophotometry (NanoDrop)
Support Protocol 3: Analysis of DNA Sequence Data
Alternate Protocol: APC Gene Sequencing Using M13 Tailed Primers
Basic Protocol 2: APC Analysis for Gross Rearrangements Using Real-Time PCR
Support Protocol 4: Data Analysis of Real Time PCR: Quantitation of Results
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: APC Gene Mutation Analysis by DHPLC and Direct Sequencing

Materials

10× FastStart PCR buffer with 2.5 mM MgCl₂ (Roche Applied Science)
dNTP mix: 2.5 mM dATP, dCTP, dGTP, dTTP (appendix 2D)
FastStart Taq DNA polymerase (Roche Applied Science; store at –20°C in the cooler container until ready for use, then return to freezer as soon as possible; mix well before use)
50 ng/µl wild-type template DNA (DNA tested in the laboratory and established not to have APC mutations, nor to be very polymorphic; isolated as in Support Protocols 1 and 2)
50 ng/µl patient template DNA (Support Protocols 1 and 2)
10 µM fragment-specific forward and reverse primers (Table 10.8.1)
DHPLC reagents (also see unit 7.10):
- DHPLC-grade H₂O
- Buffer A: 0.1 M triethylammonium acetate (TEAA)
- Buffer B: 0.1 M TEAA containing 25% (v/v) acetonitrile, pH 7.0
- Syringe washing solution: 8% acetonitrile
- 75% acetonitrile
- pUC18 plasmid, HaeIII digest, for quality-control purposes
BigDye Cycle Sequencing Reaction Kit (Applied Biosystems) including:
- Terminator Ready Reaction Mix
- 5× sequencing buffer

96-well optical PCR plate (Applied Biosystems)
Thermal cycler with heated lid
WAVE DNA Fragment Analysis System with WAVEMAKER or NAVIGATOR software v. 4.0 (Transgenomic, Inc.)
Millipore Multiscreen PCR 96-well plate
Millipore vacuum manifold
DNASep column (Transgenomic, Inc.)
96-well microtiter plates
Performa V3 96-well short plate (prepacked with gel-filtration medium; Edge Biosystems; http://www.edgebio.com/)
Centrifuge with microtiter plate carrier
ABI Prism Sequence Analysis System
Additional reagents and equipment for agarose gel electrophoresis (unit 2.7), DHPLC (unit 7.10), cycle sequencing (unit 7.7), and sequence analysis (Support Protocol 3)

Support Protocol 2: Quantitation of Genomic DNA by Spectrophotometry (NanoDrop)

Materials

Activated type XV calf thymus DNA standard (Sigma)
DNA hydration solution (Gentra)
DNA sample (Support Protocol 1) in DNA hydration solution

1.5-ml screw-cap microcentrifuge tubes with O-rings
NanoDrop ND-1000 spectrophotometer (http://www.nanodrop.com/)
Nuclease-free H₂O (Promega)

Support Protocol 3: Analysis of DNA Sequence Data

Materials

Sequences obtained via ABI Prism Sequence Analysis System (in ABI format; Basic Protocol 1)
Computer running the following:
- Microsoft Windows XP or 2000
- Mutation Surveyor software (SoftGenetics; http://www.softgenetics.com/)
- Microsoft Excel

Alternate Protocol: APC Gene Sequencing Using M13 Tailed Primers

Additional Materials (also see Basic Protocol 1)

APC M13-tailed primer sequences (Table 10.8.5)

Basic Protocol 2: APC Analysis for Gross Rearrangements Using Real-Time PCR

Materials

2× TaqMan Universal Master Mix (Applied Biosystems)
10 µM primers for real-time PCR (Table 10.8.6)
5 µM TaqMan fluorescent probe (Applied Biosystems)
20× RNase P primer/probe mix (Applied Biosystems)
10 ng/µl wild-type template DNA (Support Protocols 1 and 2)
10 ng/µl patient template DNA (Support Protocols 1 and 2)

96-well PCR plates
Optical adhesive plate covers
Centrifuge with microtiter plate carrier
ABI 7500 Real-Time PCR machine (Applied Biosystems) with computer and corresponding software

Additional reagents and equipment for real-time PCR (unit 12.13)

Table 10.8.6 APC Real-Time PCR Primer and Probe Sequences^a

Primer name	Primer sequence	Length	PCR size (bp)

APC Ex1F	GGCACTGAAGATGGAGAACTCAA	23	72
APC Ex1R	TCCAGTTTTGTAAGATGATTGGAATT	26
APC Ex1 Probe	VIC-CTTCGACAAGAGCTAGAAG-MGBNFQ	19
APC Ex2F	CAGATTGATTTATTAGAGCGTCTTAAAGG	29	115
APC Ex2R	TCGAGAGAAGCTGTACTTGGATCTAC	26
APC Ex2 Probe	VIC-CTCAAATTGTCATCTTTAGGTG-MGBNFQ	22
APC Ex3F	CTTATGGAAGCCGGGAAGGATC	22	58
APC Ex3R	ACAAACCCTCTTCTTGGAAATGAAC	25
APC Ex3 Probe	Cy5- CCGTTCTGGAGAGTGCAGTCCTGTTCCT-IAbRQ	28
APC Ex4F	GGTCATTGCTTCTTGCTGATCTT	23	73
APC Ex4R	AGATTCTGAAGTTGAGCGTAATACCA	26
APC Ex4 Probe	NED-CAAAGAAGAAAAGGAAAAAG-MGBNFQ	20
APC Ex5F	TGACCAGAAGGCAATTGGAAT	21	63
APC Ex5R	CTAGTTGTTCTTCCATCGCAACTC	24
APC Ex5 Probe	VIC-TGAAGCAAGGCAAATC-MGBNFQ	16
APC Ex6F	TTAGCGAAGAATAGCCAGAATTCA	24	71
APC Ex6R	GGGACTGTAAAAGCTGTCGTATACG	25
APC Ex6 Probe	VIC-CAAATCGAAAAGGACATACT-MGBNFQ	20
APC Ex7F	CATCTCAGAACAAGCATGAAACCG	24	61
APC Ex7R	CCATTACCAGAAGTTGCCATGTTG	24
APC Ex7	Cy5- CTCCTTGACCTTCATTCTGCCGCTCAGC-IAbRQ	28
APC Ex8F	CTACACGAATGGACCATGAAACA	23	64
APC Ex8R	CGAGGTGCAGAGTGTGTGCTA	21
APC Ex8 Probe	VIC-CCAGTGTTTTGAGTTCTA-MGBNFQ	18
APC Ex9F	TGCAGCACTCCACAACATCA	20	58
APC Ex9R	TTCACGCCTGCCTCTCTTG	19
APC Ex9 Probe	VIC-TCACTCACAGCCTGATG-MGBNFQ	17
APC Ex10F	CAGCTCCTGTTGAACATCAGATCT	24	74
APC Ex10R	TCTATGCTCTTCATCAAATGAAAGTTTC	28
APC Ex10 Probe	VIC-CTGCTGTGTGTGTTCTA-MGBNFQ	17
ACP Ex11F	TCCTCTTGCCCTTTTTAAATTAGG	24	71
ACP Ex11R	ACATTTCACAGTCCACTTGCAATAA	25
ACP Ex11 Probe	VIC-ACTACAGGCCATTGCA-MGBNFQ	16
APC Ex12F	AGGCTGCATGAGAGCACTTGT	21	71
APC Ex12R	TTCTAAATAGTACCTGCTGTAAGTCTTCACT	31
APC Ex12 Probe	VIC- CCCAACTAAAATCTG-MGBNFQ	15
APC Ex13F	TTGGCGAGCAGATGTAAATAGTAAA	25	68
APC Ex13R	ATTCCATCAATGCTTTCACACTTC	24
APC Ex13 Probe	VIC-AGACGTTGCGAGAAGT-MGBNFQ	16
APC Ex14F	TCTTACTTACCGGAGCCAGACAA	23	61
APC Ex14R	TCCGTAATATCCCACCTCCACTT	23
APC Ex14 Probe	VIC-CACTTTAGCCATTATTG-MGBNFQ	17
APC Ex15-1F	CATGTGGAACTTTGTGGAATCTCTC	25	60
APC Ex15-1R	TGAATGAATGAGGTTCTTGAGCATG	25
APC Ex15-1 Probe	Cy5- CCCCATGTCCCATAATGCTTCCTGGTCTT-IAbRQ	29
APC Ex15-8F	TGCCAACAAAGTCATCACGTAAA	23	66
APC Ex15-8R	CAGGTGGAGGTAATTTTGAAGCA	23
APC Ex15-8 Probe	VIC-CAAAAAAGCCAGCCCAGA-MGBNFQ	18
APC Ex15-14F	CAAAACTGACAGCACAGAATCCA	23	59
APC Ex15-14R	AAGACCCAGAATGGCGCTTA	23
APC Ex15-14 Probe	VIC-TGGAACCCAAAGTC-MGBNFQ	14

^aEndogenous control.

Support Protocol 4: Data Analysis of Real Time PCR: Quantitation of Results

Additional Materials (also see Basic Protocol 2)

Wild-type female DNA (Support Protocols 1 and 2)

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Figures

Figure 10.8.1

M13 tailed primer design. (A) and (B) show M13 sequences used as tails for forward and reverse PCR primers, respectively.

View Image
Figure 10.8.2

DHPLC analysis of mutations in exon 9 of the APC gene. At the T_m of 58°C, distinctive heteroduplex patterns are observed for the Y935X (trace 2), 349+1insGTAT (trace 3), R332X (trace 4), 313del2 (trace 5), Ex9+3 G>C (trace 6), as compared to the control (trace 1).

View Image
Figure 10.8.3

Real-time PCR analysis for detecting deletions in the APC gene. Real-time data of a patient sample with deletion of exon 4 of the APC gene, showing reduced cycle threshold (Ct) value compared to control sample. RNase P is used as an endogenous control for the assay. The wild-type control and patient DNA are indicated by arrows. The y-axis quantity, delta Rn, is defined as the magnitude of the signal generated by the given set of PCR conditions. The delta Rn value is determined by the formula delta Rn = (Rn⁺) – (Rn^–), where Rn⁺ is the Rn value of a reaction containing all components including the template and Rn^– is the Rn value of unreacted sample (i.e., background). In a typical real-time experiment, delta Rn undergoes several phases as in a typical polymerase chain reaction: phase 1 is baseline (little or no amplification); phase 2 is the exponential phase (template doubling every cycle); and phase 3 is the plateau phase. As a general rule delta Rn will increase with cycle number until the reaction reaches the plateau phase. The cluster of traces on the left is background.

View Image

Literature Cited

Literature Cited
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