8. METHODS

8.1. The Polymerase Chain Reaction

The polymerase chain reaction(PCR) provides a powerful technique for directly amplifying short segments of the genome, i. e. specific segments of the DNA strands. Effecting a PCR re-quires knowledge of the sequence on either side of the target region and allows amplification of a region between two defined sites.

The PCR protocol starts with denaturation of the DNA preparation at 94°/95° (generally an extract of the whole genome). The double-stranded DNA is separated by heat into single-stranded DNA serving as a template for amplification. Amplification of the DNA is achieved by DNA polymerase producing a complementary DNA-strand from the 5´OH end to the 3´OH end. As a starting point DNA polymerase needs a double-stranded sequence of DNA. To produce this double-stranded DNA the single-stranded DNA is annealed with two short primer sequences (20 bases each), one forward an one reverse primer. Each primer is complementary to a site on the opposite strand determining the target region (up to 2 kb). The reaction cocktail contains the two primers, the template DNA, thermostable DNA-polymerase (taq DNA-polymerase), all four 2`-deoxynucleoside 5`- triphosphates (dNTPs) dATP, [page 28↓]dCTP, dGTP, dTTP, a buffer and magne-sium chloride ions. After denaturation the temperature is lowered in a second step (annealing) to 55°-57° so both primers can ideally anneal to their complementary regions on the template DNA. In a third step (extension) the temperature is raised to 72°, the temperature optimum for the taq DNA-polymerase, and new DNA-strands are synthesized complementary to the template DNA. The entire cycle is repeated 25-32 times resulting in copies of non-determined length (with only one primer at one end) but also copies with a length defined by the two primers. Throughout amplification the number of copies of non-determined length grows linearly whereas the number of copies of determined length grows exponentially. Therefore only products of determined length exist after 25-32 cycles. The number of copies of the target sequence practically doubles with each cycle until reaching a plateau at which more primer-template accumulates than the enzyme manages to amplify during the cycle. A given target sequence may be amplified 4 x 106 times in 25 cycles. At this point the number of target product no longer increases exponentially. The PCR is accomplished in programmable incubation blocks which guaranty quick and precise temperature changes. The availability of thermostable taq-DNA polymerase from a thermophilic bacterium, able to withstand even multiple denaturation steps at 95° without losing all of its activity made automatization of the PCR possible. Before that, DNA polymerase had to be added after each denaturation step.

This method provides a powerful possibilitiy to investigate individual alleles and potential candidate genes involved in a disease. PCR is as sensitive as to genotype a single cell, offering analysis of a circumscribed cell population, f. ex. spermatozoa, but also amplification of rather small tissue material.

8.2. The restriction to PCR sensitivity: Genotyping errors caused by taq DNA polymerase

• A potential source of genotyping errors is contributed to unspecific annealing, i.e. non-templated addition of a single nucleotide, predominantly adenosine, to the 3´ OH end of the DNA strand by taq DNA polymerase. Taq DNA polymerase is marker-specifically catalyzing the amplification of microsatellite loci, however, experimental variation of the frequency of adenosine addition is often difficult to avoid. The likelihood with which a marker undergoes "+A" (+ adenosine) modification also is marker specific but the factors promoting this phenomenon have not been defined. Allelic misidentification is commonly generated by incorrect labelling of spurious noise peaks or peaks one nucleotide greater in size than the true allele. Consequently, genotyping errors occur and the same allele may be idetified as the true allele in some family members and as the product one nucleotide greater than the true allele in other members. But even an allele in a single individual can be identified inconsistently in repeated amplifications or electrophoreses. One possibility to compensate for unspecificity (incorrect products) is by determining the exact size of the products, e.g. by computer-based analysis (e.g. GENESCAN).

• Using PCR technique, one must compromise between a low temperature which gives a high amount of products but also a lot of unspecific annealing, and a high temperature which gives high specificity but little products. The extent of range within one can modulate temperature depends on primer design and content of GC basepairs in the target sequences. Thus, another approach to decrease the error rate in PCR is the modification of thermocycling protocols to dinucleotide repeat markers to avoid problematic partial modification. A first protocol cycles between denaturation and anealing leaving out both the extension step to each cycle and the final extension step, thus diminuishing the degree of non-templated nucleotide addition by taq DNA polymerase and generating more product, a second protocol lengthens the final extension period to 90 minutes so that the enzyme catalyzes non-templated nucleotide addition to it´s maximum. Alternatively, a thermostable taq DNA polymerase version lacking all "+A" activity might be used but which is currently not available (Smith, Carpten et al. 1995).

Preferential PCR amplifying implies that when getting close to the plateau phase of the amplifying process or when having a for short elongation phase shorter products are amplified relatively more effectively and maybe more rare porducts as well, due to diminished dimerisation of products.

• A dinucleotide repeat and its "stutter bands" (one to two peaks characteristically smaller in size and in peak height) can be detected in the form of a ladder of peaks seperated by one nucleotide as a result of partial "+A" and partial true allelic detection. It is inferential that a dinucleotide repeat microsatellite marker modified to a 50% degree (probably due to "polymerase slipping") would imply the greatest potential for error. One way of lowering the error rate in genotyping would be the [page 29↓]substitution of tri- and tetranucleotide repeats for dinucleotide repeats. Tri- and tetra nucleotide repeats present with more faint or absent stutter bands. On the other hand, fewer markers may be multiplexed (scored) per gel due to greater allele size range of tri- and tetranucleotide repeat markers. In addition, dinucleotide repeats provide the advantage of their prominent stutter pattern, on the contrary minimal for tetranucleotide repeats, quite helpful in distinguishing noise and background peaks from true alleles. Furthermore, a higher number of dinucleotide repeats have been identified throughout the genome. These characteristics demand development of further methods for their optimized use.

8.3. DNA extraction and PCR amplification

The tumors were snap frozen at the time of surgery, then cryosections were made from each tumor sample and tumorous tissue from each patient was identified on a Hematoxylin-Eosin stained slide. This slide served as a road map to process the tumor tissues into one Eppendorf tube each. Lesions with a low proportion of contaminating fibroblasts were selected for analysis. For tumors 5962 and 5807 the cryosections were microdissected in order to avoid gross con-tamintion by non-tumorous cells. From these frozen tissues DNA was extracted by standard proteinase K/SDS digestion and phenol extraction. Paired germline DNA was extracted from leucocytes with Wizard Genomic DNA Purification Kit (Promega) or normal intestine tissue.

DNA extraction from tumor tissues

On day one, according to the size of the tumor sample, 10 to 30 microdissected 20 µm slices were processed into one Eppendorf-tube filled with 450 µl SE-buffer (15 ml 5M NaCl, 50 ml 0,5M EDTA, 30 ml 1M Tris pH 8,0 and double-destilled water to a total of 1000 ml, then autoclaved). 10 µl proteinase K (20 µg/ml), 25 µl 10% SDS and 1 µl RNAse A (10 µg/ml) were added and all was incubated over night at 37°. On day two, 1 volume phenol/chisam (1:1) was added, carefully shaken and vortexed. Then, the extraction mixture was centrifuged for 3 minutes at 14000 rpm in room temperature. The upper phase was processed into a new Eppendorf-tube, the phenol/chisam extraction was repeated and the upper phase transferred to yet another Eppendorf-tube. 3M NaAc, ph 5,2 were added to a final concentration of 0,3 M NaAc, after which one volume Isopropanol was added. All was mixed carefully untill white strands of DNA precipitated. The DNA was centrifugated for 15 minutes at 14000 rpm at room temperature. The water phase was removed with a Pasteur pipet and the DNA pellet was rinsed with 70% ethanol and air-dried for 10 minutes under warm light. The DNA was then dissolved in 100 µl TE-buffert (1 ml 1M Tris pH 7,9, 200 µl 0,5M EDTA and sterile water to a total of 100 ml). The DNA was left standing over night and on day three a test gel was run in order to check the degree of DNA degradation and to get a preliminary DNA concentration measurement. DNA concentration was then attained by density (OD) measurement.

DNA extraction from leucocytes:

(Please, refer to Wizard´s protocol "DNA-extraction from leucocytes"). 900 µl of cell lysis solution were added to a sterile 1,5 ml Eppendorf-tube. The tube of patient blood was gently rocked until thoroughly mixed and 300 ml of blood were transferred into the tube containing the cell lysis solution. the tube was inverted 5-6 times to mix and incubated for 10 minutes at room temperature to lyse the red blood cells. Then, the tube was centrifuged for 20 seconds at 12000 rcf at room temperature. As much supernatant as possible was removed and discarded without disturbing the visible white pellet. The tube was vigorously vortexed until the white bloodcells were resuspended. 300 µl of nuclei lysis solution were added to the tube and the content pipetted several times to lyse the cells. 100 µl of protein precipitation solution were added to the nuclear lysate and vortexed vigorously for 10-20 seconds. The sample was then centrifugated for 3 minutes at 12000 rcf at room temperature until a dark brown pellet was visible. The supernatant was transferred to a clean 1,5 ml Eppendorf-tube containing 300 µl of isopropanol. The solution was mixed by inversion until the white thread-like strands of DNA formed a visible mass. The tube was centrifugated for 5 minutes at 12000 rcf at room temperature until the DNA was visible as a white pellet. The supernatant was decanted and 300 µl of room temperature 70% ethanol were added to wash the DNA, then the tube was centrifugated for one minute at 12000 rcf at room temperature. The ethanol was aspirated with a Pasteur pipet, the tube inverted on clean ab-sorbent paper and the DNA pellet air-dried for 10-15 minutes. Finally, 100 µl of [page 30↓]DNA hydration solution were added to the tube and the DNA rehydrated by incubating at 65° C for one hour. DNA was stored at 4° C.

PCR amplification

Paired tumor and non-tumor DNA from the same patient served as a template for PCR ampli-fications. Two sets of oligonucleotide primers were used, microsatellite markers (simple repeated sequences of DNA, mono-, di-, tri- and tetranucleotide repeats) of the first set were obtained from the department of clinical genetics at Uppsala University hospital. These primers are ampli-fying the polymorphic loci in the human genome as defined in the screenig set 6 released by the Cooperative Human Linkage Center (CHCL) in the U.S.. The second set was purchased from Research Genetics, Inc., U.S. These primers are amplifying markers within the human linkage map as defined in the screening set 9A from CHCL. For both sets the forward markers are labelled with a fluorescent dye, either 6-FAM (blue), HEX (yellow) or TET (green) to be ana-lyzed on the ABI PRISM Genetic Analyzer. PCR reactions were performed in an ABI PRISM 877 thermal cycler (Perkin Elmer Applied Biosystems). For primers from the first set the PCR reactions contained 10-20 ng of template DNA, 2-6 pmol of each forward and reverse primer, 0,2 mM each dNTP (2´-deoxynucleotide 5´- triphosphate) (Life Technologies, Inc.), 1x PCR buffer, 1,5 mM Magnesium Chloride (Life Technologies, Inc.), 0,5 units Taq DNA Polymerase (Life Technologies, Inc.) and autoclaved distilled water to a final volume of 5-10 µl. Cycling was achieved as follows: denaturation at 95° for 3,5 minutes, followed by 30 cycles of de-naturation at 95° for 30 seconds, annealing at 55° for 30 seconds and extension at 72° for 30 seconds. A ten-minute final extension at 72° was carried out to finish the amplification. PCR re-actions for markers from the second set contained 10 ng of template DNA, 1,2 pmol of each for-ward and reverse primer, 0,2 mM each dNTP (2´-deoxynucleotide 5´- triphosphate) (Life Technologies, Inc.), 1x PCR buffer, 1,5 mM Magnesium Chloride (Life Technologies, Inc.), 0,5 units Taq DNA Polymerase (Life Technologies, Inc.) and autoclaved distilled water to a final volume of 5 µl. Cycling was performed by a denaturation step ar 95° for 2 minutes, 30 cycles of denaturation at 94° for 45 seconds, annealing at 56° for 45 seconds and extension at 72° for 60 seconds. A six-minute final extension at 72° was carried out to finish the amplification. Different temperatures and times for the different steps of amplification were chosen according to re-commendation by the authors of the screening set 9 edition by CHLC.

8.4. LOH screening

After cycling, 1µl of GENESCAN TAMRA lane standard (Perkin Elmer Applied Biosystems) and 17µl of Formamid were added to a screening pool consisting of 1-4µl of each of 5-8 micro-satellite PCR products of a given panel (markers grouped together according to their size) to be pooled together and coelectrophoresed unambiguously. Then, the screening volumes (27-37µl PCR reaction) were denaturated at 95° for 5 minutes and transferred to an ABI PRISM 310 Genetic Analyzer (Perkin Elmer Corporation) consisting of a laser-induced fluorescence capillary electrophoresis instrument and a Macintosh computer including "Genescan Perkin-Elmer Corporation" software for data collection and analysis of fluorescent-labelled DNA frag-ments for size and quantification. Each sample was loaded on Performance Optimized Polymer 4 (POP4) and the products separated by electrophoresis through the capillary at 15 kV electro-phoresis voltage, 9 µA electrophoresis current, laser power of 9,9 mV and 60° for 24 minutes. The light intensities of each product were stored as electric signals and displayed in the form of coloured peaks (one peak representing one allele) and the peak amplitudes were analyzed.

Heterozygosity, i.e. the presence of two distinct alleles in normal tissue has been the essential requisite for evaluation of LOH. Decreased peak amplitude of either tumor allele in hetero-zygous individuals was calculated in relation to peak amplitudes of paired normal DNA.

A reduction of the relative amplitude of 40% or more (a retention of 60% or less, respectively) was considered LOH. Given the peakheights of two of different-sized alleles in non-tumorous DNA in heterozygous individuals (N1 and N2) and of loss of one allele of the corresponding tumorous DNA (T1 and T2) the retention level was calculated as follows:

Retention level = (T 1 /T 2 )/(N 1 /N 2 ).


[page 31↓]

72 fluorescent microsatellite markers from the first set and 64 markers from the second set as well as two custom-made 11q13 primers, in total, 131 different mikrosatellite markers were used to genotype DNA from the eight midgut carcinoids. The markers were distributed over the entire genome exept for chromosomes X and Y, with at least two markers per chromosomal arm. The microsatellite markers used in the analysis are listed beneath.

Fluorescent microsatellites used in our genome-wide screening for LOH in midgut carcinoid tumors

custom-made primers ( Perkin Elmer Corp.)

PYGM

INT2

Weber screening set 6 (Nordic Consortium Primer Resource Center at the department of Clinical Genetics, Uppsala, Sweden

D1S1622, D1S551, D1S1589, D1S549,

D2S1356, D2S1649, D2S434,

D3S2387, D3S1768, D3S2427, D3S2398,

D4S2639, D4S2397, D4S2408, D4S2368, D4S2431,

D5S2505, GATA7C06, D5S2501, D5S816,

D6S1281, D6S1009, D6S1003, D6S1277,

D7S513, D7S1802, D7S821, D7S1804,

D8S1099, D8S592, D8S1179, D8S373,

D9S925, D9S1118, D9S302,

GAAT5F06, D10S1239,

D12S374, D12S391, D12S373, GATA32F05,

D13S173,

D14S749, D14S611, D14S118,

D15S652, D15S642,

D16S748, D16S769, D16S2624,

D17S1308, D17S1298, D17S1299, D17S809, D17S1290,

D18S843, D18S64, D18S541,

D19S247, GGAT2H06, D19S601,

D20S95, D20S604, D20S481, D20S1085,

D21S1435, D21S1270, D21S156, D21S1446,

D22S685, D22S683, D22S445

[page 32↓]

Weber screening set 9 ( Genetic Research Inc.)

D1S1612, D1S552,

GATA165C07, D2S1356, D2S1394, D2S139,

D3S2387, GATA164B08,

D4S2639, D4S2431, D4S1652,

D5S2488, D5S807, GATA134B03, D5S2500, D5S1505, D5S820, D5S1456,

GATA163B10, FA3A1, D6S1053,

GATA137H02

GATA62F03, D9S925, D9S910, D9S934, D9S1838,

D10S1435, D10S1430, D10S1426, D10S677,

D11S1999, D11S1392, D11S1984, D11S2000,

D12S391, D12S1042, PAH, D12S395,

D13S317, D13S285,

D14S617, GATA136B01,

D15S643, D15S657,

ATA41E04, D16S764, D16S753, D16S3253, D16S2624, D16S539,

D17S1293,

D18S481, D18S877, D18S858, D18S844,

D19S433,

D20S482, D20S470, D20S481, D20S480, D20S171,

GATA188F04

Smad4/DPC4 analysis

In order to investigate a possible role of the TSG SMad4/DPC4, located on 18q21, in the neo-genesis of our tumors, sequence analysis of exon 8-11 was performed. Only exon 8-11 were ana-lysed since these exons are were most often mutated in previously investigated tumors (Bartsch et al. 1999).

All tumor samples underwent PCR amplification using oligonucleotide primers flanking exon 8-11. The amplified samples were subjected to semiautomated sequencing on ABI 310 using ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit with Ampli Taq DNA Polymerase FS (Perkin Elmer Corp.).

Immunohistochemical staining with monoclonal antibodies to Smad4/DPC4 (clone B8, Santa Cruz) was additionally performed on all tumors exept for 5216. The latter tumor was excluded due to lack of tissue material.


[page 33↓]

Figure 1.Examples of LOH in tumor T5216. Extensive LOH at chromosome 18q
(marker D18S844). Partial LOH at chromosome 4p (marker D4S2639). No LOH at
chromosome 11q (marker D11S1984). LOH revealed by allele reduction of one allele
in the tumor tissue.


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