Nuclear Medicine Resources Manual

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CHAPTER 5. GUIDELINES FOR GENERAL IMAGING

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(3) Detection of mutations that are diagnostic for neoplasms (leukaemia),

which define prognosis (p53) and which are resistance indicators (p53).

The most common molecular approach can be used for the following

cancers:

—Bladder cancer;

—Breast cancer;

—Colon cancer;

—Leukaemia;

—Liver cancer;

—Lymphoma;

—Melanoma;

—Multiple endocrine neoplasia;

—Neuroblastoma;

—Ovarian cancer;

—Prostate cancer;

—Thyroid cancer.

5.13.4.1. The p53 genetic marker

The most common genetic tumour marker is p53. Mutations in this gene

and loss of the normal allele is the most common alteration that leads to the

progression of cancer. These mutations can be determined by PCR and

sequencing as well as by other techniques, such as single strand conformation

polymorphism (SSCP), which are directed towards the detection of new

mutations. The p53 gene is involved in distinct neoplasms, and alterations in

this gene are considered as diagnostic mutations, prognostic markers, a suscep

tibility indicator or a resistance indicator.

Neoplasms in which p53 is involved include:

—Bladder tumours;

—Breast tumours;

—Colorectal tumours;

—Head and neck tumours;

—Leukaemia;

—Liver tumours;

—Lymphoma;

—Ovarian tumours;

—Prostate tumours.

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The early appearance of breast cancer (at a mean age of 37 years) is

highly associated with p53 mutations. Fifty per cent of Li–Fraumeni syndrome

cases, a predominantly inherited syndrome associated with multiple primary

neoplasms of children and young adults, have a germ line p53 mutation and

49% of families with germ line p53 mutations meet the criteria for Li–Fraumeni

syndrome: occurrence of sarcoma before 45 years of age and at least one first

degree relative with a tumour before 45 years of age or a sarcoma at any age.

Inherited germ line p53 mutations are associated with breast cancer

(24%), bone sarcomas (13%), brain tumours (12%) and soft tissue sarcomas

(12%). Adrenocortical carcinoma is not common (4%) but in children is

almost always associated with a germ line p53 mutation.

Another interesting feature is found in some geographical areas where an

overlapping of hepatitis B infection and exposure to the mould toxin (aflatoxin

B) leads to mutation in the p53 gene. This can be reflected in the future

appearance of an aggressive hepatocellular carcinoma.

The p53 gene can be considered a prognostic marker for several tumours.

In colorectal carcinoma p53 mutations are present in 75% of cases. In lymph

node negative breast cancer the 8 year survival rate in p53 negative women is

82%, whereas in p53 positive cases it is 66%. Furthermore, in lymph node

positive cases, p53 positive cases are associated with a 20% 8 year survival rate

and p53 negative cases present a 56% survival, leading to the conclusion that

the presence of mutations in the gene corresponds to a poor overall survival. In

prostate cancer, p53 is considered to be a marker of tumour progression. In

lymphoma, the survival is lower in patients bearing mutations in the p53 gene

than in those patients with wild type p53. Considering colorectal carcinoma, the

5 year survival rate in p53 positive patients is 18% lower than that of p53

negative patients.

Aggressive myeloid chronic leukaemia presents mutations in p53 in 29–

50% of cases. These values contrast to those found in typical cases (6–9%).

Furthermore, the survival time in p53 positive patients with that kind of

leukaemia is 12–18 months, while in p53 negative patients the survival period

extends to 60–117 months.

Resistance to a specific therapeutic scheme can also be affected by

mutations in the p53 gene. In breast cancer, p53 mutations increase resistance

of tumours to ionizing radiation. In B cell chronic leukaemia, the mutations in

the gene are translated as a resistance to chemotherapy. The same

phenomenon is observed in ovarian cancer, where wild type p53 tumours are

more sensitive to chemotherapy.

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5.13.5. Basic protocols in molecular biology

5.13.5.1. Isolation of DNA

DNA is usually isolated by digestion of cells with proteinase K in the

presence of EDTA and a detergent such as SDS, followed by extraction with

phenol. This method yields DNA whose size (100–150 kb) is adequate for

Southern Blot analysis.

DNA from biopsies (tissue) should be immediately processed or placed at

–20°C. Commercial kits for purifying genomic DNA from fresh tissue biopsies

or from archival material are available. Special protocols for paraffin

embedded tissue are provided and should be strictly followed. Removal of the

paraffin is recommended before extraction of the DNA.

If it is necessary to extract DNA from blood or buffy coat, the use of

commercial kits is also recommended. For this specific procedure, the vast

majority of available kits require the use of 500 mL of whole blood/EDTA or

buffy coat. The results of manipulations of DNA extracted with commercial

kits are usually better than those with home-made traditional protocols.

In field work where biopsies are taken, one of the simplest and perhaps

best methods of preserving the DNA is to place the sample in 50% ethanol.

Although DNA can be extracted from formalin fixed tissues, the least degraded

DNA and the highest yield of DNA can be isolated from ethanol fixed samples.

The fixed cells are suitable for DNA extraction for many days when left at

room temperature and for more than 6 years at 4ºC. Thus, this protocol is ideal

for sample preservation by field molecular biologists. In animal blood samples,

citrated blood can be used for DNA analysis after 1 or 2 days at room temper

ature, perhaps longer. A good way to preserve blood samples for DNA analysis

is to simply make air-dried blood smears on slides or use filter paper.

5.13.6. Polymerase chain reaction (PCR) technology

5.13.6.1. Optimization of the PCR method

The primers used in the PCR hybridize to opposite strands of the target

sequence and are oriented so that DNA synthesis by the polymerase takes

place across the region between them. Since the extension products are also

complementary to and capable of binding primers, successive cycles of amplifi

cation essentially double the amount of the target DNA synthesized in the

previous cycle. The result is an exponential accumulation of the specific target

fragment by a factor of approximately 2

, where n is the number of cycles of

amplifications performed.

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423

The PCR is so sensitive that a single DNA molecule can be amplified, and

single copy genes are routinely extracted out of complex mixtures of genomic

sequences and visualized as distinct bands on agarose gels. The use of

thermostable DNA polymerases and automation of the method invented by

Mullis in 1987 have led to the development of diverse PCR applications.

Each new PCR is likely to require optimization, especially when used for

routine diagnostic or analytical procedures in which optimal performance is

necessary. Problems that can be encountered are:

—No detectable product or a low yield of the desired product;

—The presence of non-specific background bands due to mispriming or

misextension of the primers;

—The formation of ‘primer-dimers’, which compete for amplification with

the desired product;

—Mutations or heterogeneity due to misincorporation.

5.13.6.2. Standard PCR amplification protocol

Standard conditions will amplify most target sequences and are presented

to provide starting conditions for designing new PCR applications.

First of all a 50 mL reaction is set up in a microfuge tube (adequate for the

available thermocycler). If the PCR machine is not supplied with a hot bonnet,

a mineral oil overlay will be necessary to prevent evaporation:

(a) 20 pmol of each primer.

(b) For the buffer 20mM Tris-HCl (pH8.3), 1.5mM MgCl

25mM KCl, 0.05%

Tween 20 and 100 mg/mL of BSA is used.

(d) Two units of Taq DNA polymerase.

(e) A DNA template (10

–10

target molecules).

(f) Perform 25–35 cycles of PCR using the following temperature profile:

—Denaturation: 96ºC for 15–30 s (a longer initial time of 4 min is usually

desirable).

—Primer annealing: 55ºC for 30 s.

—Primer extension: 72ºC for 30 s to 1 min.

Cycling should conclude with a final extension at 72ºC for 5 min.

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(a) Enzyme concentration

The recommended concentration range for Taq DNA polymerase

(PerkinElmer Cetus) is between 1 and 2.5 units per 100 mL reaction when the

other parameters are optimum. However, enzyme requirements may vary with

respect to individual target templates or primers. When optimizing a PCR, it is

recommended to test enzyme concentrations ranging from 0.5 to 5 units per

100

mL and to assay the results by gel electrophoresis. If the enzyme

concentration is too high, spurious non-specific background products may

accumulate, and if it is too low, a low yield of products will be in evidence. As

soon as the enzyme is received it should be aliquoted into 10 mL samples and

stored at –20°C in Area 1 (Section 3.8).

(b) Deoxynucleotide triphosphates

Stock dNTP solutions should be 10mM, aliquoted and stored at –20ºC in

Area 1 (Section 3.8). Working stock solutions each containing 2mM dNTP are

recommended. Deoxynucleotide concentrations between 50 and 200mM each

result in an optimal balance of the yield, specificity and fidelity. The four

dNTPs should be used at equivalent concentrations to minimize misincorpo

ration errors. Low dNTP concentrations minimize mispriming at non-target

sites and reduce the likelihood of extending misincorporated nucleotides. The

lowest dNTP concentration appropriate for the length and composition of the

target sequence should be decided upon.

It is beneficial to optimize the magnesium ion concentration. The

magnesium concentration may affect all of the following: primer annealing,

strand dissociation temperatures of both template and PCR product, formation

of primer-dimer artefacts, and enzyme activity and fidelity. Taq DNA

polymerase requires in addition free magnesium bound by template DNA,

primers and dNTPs. Accordingly, PCRs should contain 1.0–5mM MgCl

(d) Other reaction components

A recommended buffer for PCR is 10–50mM Tris-HCl (between pH8.3

and 8.8). Up to 50mM of KCl can be included in the reaction mixture to

facilitate primer annealing. However, values above 50mM inhibit Taq DNA

polymerase activity. Bovine serum albumin (100 mg/mL) and non-ionic

5.13. MOLECULAR METHODS

425

detergents such as Tween 20 are included to help stabilize the enzyme, although

many protocols work well without added protein.

(e) Primer annealing

The temperature and time required for primer annealing depend upon

the base composition, length and concentration of the amplification primers.

An applicable annealing temperature is 5ºC below the true melting

temperature (T

) of the amplification primers. Because Taq DNA polymerase

is active over a broad range of temperatures, primer extension will occur at low

temperatures, including the annealing step. The range of enzyme activity varies

by two orders of magnitude between 20 and 85ºC. Annealing temperatures in

the range of 55–72ºC generally yield the best results. At typical primer concen

trations (0.2mM), annealing will require only a few seconds. Increasing the

annealing temperature enhances discrimination against incorrectly annealed

primers and reduces mis-extension of incorrect nucleotides at the 3¢ end of

primers.

(f) Primer extension

Extension time depends upon the length and concentration of the target

sequence and upon temperature. Primer extensions are traditionally performed

at 72°C because this temperature is near optimal for extending primers.

Estimates for the rate of nucleotide incorporation at 72ºC vary from 35 to 100

nucleotides per second, depending upon the buffer, pH, salt concentration and

nature of the DNA template. An extension time of 1 min at 72°C is considered

sufficient for products up to 2 kb in length.

(g) Denaturation time and temperature

The most likely cause for failure of a PCR is uncompleted denaturation of

the target template and/or the PCR product. Typical denaturation conditions

are 95°C for 30 s, but higher temperatures may be appropriate, especially for

G+C-rich genomes. It only takes a few seconds to denature DNA at its strand

separation temperature; however, there may be a lag time involved in reaching

the desired temperature inside the reaction tube. Denaturation steps that are

too high and/or too long lead to unnecessary loss of enzyme activity. The half-

life of Taq DNA polymerase activity is more than 2 hours, 40 min and 5 min at

92.5, 95 and 97.5ºC, respectively.

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(h) Cycle number

The optimum number of cycles will depend mainly upon the starting

concentration of target DNA when other parameters are optimized. A

common mistake is to execute too many cycles, which can increase the amount

and complexity of non-specific background products. Of course, too few cycles

give a low product yield. Thirty cycles seems to be a good choice for a PCR

protocol.

(i) Primers

Primer concentrations between 0.1 and 0.3mM are generally optimal.

Higher primer concentrations may promote mispriming and accumulation of

non-specific product and may increase the probability of generating a template

independent artefact termed a primer-dimer. Non-specific products and

primer-dimer artefacts are themselves substrates for PCRs and compete with

the desired product for enzymes, dNTPs and primers, resulting in a lower yield

of the desired product.

(j) The Southern Blot technique

This technique refers to the transfer of DNA to fixed supports, such as

membranes. It was first developed by E.M. Southern and thus bears his name.

A specimen of DNA is digested with restriction enzymes, resulting in DNA

fragments that are then submitted to agarose gel electrophoresis. The double

stranded DNA present in the agarose gel is denatured and the electrophoreti

cally separated fragments immobilized on an inert support (a nylon membrane)

in such a way that self-annealing is prevented and yet bound sequences are

available for hybridization with an added nucleic acid probe. An adaptation of

the method can be used for amplified products from PCR where the simple

transfer of the PCR products from an agarose gel to a nylon membrane is also

named a Southern Blot experiment.

The transfer is mediated by soaking gel in NaOH 0.5N/NaCl 1.5M for

min, then in Tris-HCl 0.5M pH8.0/NaCl 1.5M for 30 min and transferring by

capillarity overnight in 20X SSC (a sodium chloride–sodium citrate buffer).

(k) Dot blot

Dot blot is a rapid technique used to detect the presence of a specific

DNA in a specimen. Dots or spots of the DNA containing sample are placed

onto a nylon membrane and fixed. Dot blot hybridization is ideally suited to

5.13. MOLECULAR METHODS

427

the analysis of multiple samples. The technique has the added advantage that it

is easy to prepare replicate filters, allowing many filter-bound sequences to be

analysed at the same time, for example with different probes or under different

hybridization and washing conditions. Multiple DNA samples are spotted next

to each other on a single filter in dots of uniform diameter. For quantitative

analysis, known amounts of DNA are applied. To evaluate the extent of hybrid

ization of the probe, a standard consisting of a dilution series of DNA dots is

applied in an identical way to the same filter. The procedure binds samples

quickly so that many samples can be handled at once. As little as 1–3 pg of a

hybridizing DNA sequence can be detected. Dot blots do not distinguish

between the number and size of the molecules hybridizing, so the hybridization

‘signal’ is the sum of all sequences hybridizing to the probe under the

conditions used. Commercial apparatus has been developed for binding

multiple samples of DNA to filters.

The DNAs (100 mL = 10 mL of DNA + 90 mL of double distilled water) are

denatured by heating for 5 min at 95°C and put on ice. Ten microliters of NaOH

4N are added and the mixture applied to a dot blot apparatus following the

manufacturer’s instructions.

(l) Binding of nucleic acid to filters

There are several types of filter currently in use for the immobilization of

DNA and ribonucleic acid (RNA), for example nitrocellulose, nylon and

chemically activated papers. The material of choice depends on the purpose of

the experiment. Nylon membranes, due to their higher resistance, are now the

most commonly used type for Southern and dot blots. For DNA dot blots,

filters with a pore size of 0.45 mm are used for large nucleic acid molecules and

0.22 mm for molecules of less than 500 nucleotides.

Ideally the binding of nucleic acids to membranes should be covalent

mediated. This will prevent the gradual leaching-off of the nucleic acids from

the surface when filters are hybridized for long periods, particularly at high

temperature. The most commonly used technique for binding of nucleic acid to

filters is that with UV light. The same goal can be achieved by baking the

membrane at 80°C for 2 hours.

5.13.6.3. Hybridization strategy

Nucleic acid hybridization, the formation of a duplex between two

complementary nucleotide sequences, is the basis for a range of techniques

now in widespread use in modern biology. Optimal use of this molecular

method requires a clear understanding of its principles so that the basic

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techniques can be modified to suit the particular purposes and conditions of a

desired experiment.

The process which underlies all of the methods based on molecular

hybridization relies on the complementarity of the two sequences involved in

duplex formation: the target DNA and the probe used. The specificity of a

hybridization assay depends on the stringency that usually is mediated by the

temperature and sodium concentration (SSC) of the hybridization and washing

solutions. The higher the temperature the more stringent is the hybridization

condition. On the other hand, as the salt concentration is decreased, the

stringency increases.

There are various types of hybridization commonly in use, such as filter

hybridization and in situ hybridization. The membrane containing the

denatured DNA is hybridized with an added nucleic acid probe. To facilitate

analysis, the probe is labelled, often with

P. Hybridization is followed by

extensive washing of the filter to remove unreacted probe. Detection of hybrids

is usually by autoradiography or a phosphoimager analyser. The procedure is

widely applicable, being used for Southern Blot and dot blot hybridization for

example.

(a) Types of nucleic acid probes for hybridization

In theory, any nucleic acid can be used as a probe provided that it can be

labelled with a marker that allows identification of the hybrids formed. In

practice, double and single stranded DNAs are mainly used as probes. The

choice of probe depends on three factors: the hybridization strategy, the availa

bility or source of material for use as a probe and the degree to which it can be

labelled. Probes can be made by cloning, PCR or using a DNA synthesizer.

(b) Labelling nucleic acids for use as probes

(i) Choice of radionuclide

Traditionally, filter hybridizations have been carried out with

radioactively labelled probes, and several radionuclides can be used in

molecular biology experiments (Table 5.22). However, for nucleic acid

hybridization,

P is the isotope of choice since its high energy results in short

scintillation counting times and short autoradiographic exposures. Each of the

four deoxyribonucleotides is available in an a-

P labelled form, of high or low

specific radioactivity, suitable for incorporation into DNA using one of the

polymerase reactions, replacing a proportion of the nucleotides in a nucleic

acid with

P derivatives. In addition, g-[

P]–ATP is also available for 5¢ end

5.13. MOLECULAR METHODS

429

labelling DNA using polynucleotide kinase, which adds

P to the end of the

molecule. Phosphorus-32 has the advantage over other radionuclides in that

high specific activities can be readily attained. Much of the technology of

filter hybridization has been developed with it. However, precautions must

be taken when handling

P because of the radiation emitted (Chapter 2).

Detection by autoradiography, while sensitive, may take a long time if there

are few counts in the hybrids. Alternatively, phosphoimagers may be used.

Furthermore, since

P has a half-life of 14.3 days, experiments should be

completed within one half-life.

(ii) Radionuclide labelling methods

There are three radionuclide labelling methods:

(1) End labelling with T4 polynucleotide kinase. Polynucleotide kinase is mostly

used to label probes for hybridizations and primers for PCR reactions, trans

ferring the g phosphate of ATP to a free 5´ OH group in the DNA. Specific

activities of 5 × 10

counts/(min·pmol) can be achieved. This method is

suitable for probes and primers that are single stranded and short (20–30

bases).

(2) Synthesis of labelled DNA probes using random oligonucleotide primers.

DNA probes can be radiolabelled to very high specific activities (5 × 10

–

4 × 10

counts/(min·mg)) synthesized in this way, by using a-

P–dNTP and

three unlabelled dNTPs as precursors. Commercial kits of random

primers are available by synthesizing on an automated DNA synthesizer,

a population of octamers that contains all four bases in every position.

The most widely used DNA polymerase for this purpose is the

Klenow fragment of E. coli DNA polymerase. This method is used for

labelling PCR products, cloned DNA molecules that are double stranded

and mostly for products larger than 100–1000 bp.

TABLE 5.22. ISOTOPE, AVAILABLE REAGENT AND ASSAY IN

WHICH RADIONUCLIDES CAN BE USED

Isotope Reagent Assay

P a-

P–dNTP Double strand DNA labelling

P–dNTP 5¢ end-labelling of single strand DNA

(oligonucleotides — probes and

primers)

P a-

P–dNTP Double strand DNA labelling