Restriction Mapping of DNA Fragments

 Restriction mapping involves the size analysis of restriction fragments produced by several restriction enzymes individually and in combination. The principle of this mapping is illustrated in Figure 1, in which the restriction sites of two enzymes, A and B, are being mapped. Cleavage with A gives fragments of 2 and 7 kb from a 9 kb molecule, hence we can position the single A site 2 kb from one end. Similarly, B gives fragments of 3 and 6 kb, so it has a single site 3 kb from one end; but it is not possible at this stage to say if it is near to A’s site, or at the opposite end of the DNA. This can be resolved by a double digestion. If the resultant fragments are 2, 3 and 4 kb, then A and B cut at opposite ends of the molecule; if they are 1, 2 and 6 kb, the sites are near each other. Not surprisingly, the mapping of real molecules (which possess many different restriction sites) is rarely as simple as this, and bioinformatic analysis of the restriction fragment lengths is usually needed to construct a map.

Fig1. Restriction mapping of DNA. Note that each experimental result and its interpretation should be considered in sequence, thus building up an increasingly less ambiguous map.

Nucleic-Acid Blotting Methods

Electrophoresis of DNA restriction fragments allows separation based on size to be carried out; however, it provides no indication as to the presence of a specific, desired fragment among the complex sample. This can be achieved by transferring the DNA from the intact gel onto a piece of nitrocellulose or nylon membrane placed in con tact with it. This provides a more permanent record of the sample since DNA begins to diffuse out of a gel that is left for a few hours. First, the gel is soaked in alkali to render the DNA single stranded. It is then transferred to the membrane so that the DNA becomes bound to it in exactly the same pattern as that originally on the gel. This transfer, named a Southern blot after its inventor Ed Southern, can be performed electrophoretically or by drawing large volumes of buffer through both gel and mem brane, thus transferring DNA from one to the other by capillary action (Figure 2). The point of this operation is that the membrane can now be treated with a labelled DNA molecule, for example a gene probe. This single-stranded DNA probe will hybridise under the right conditions to complementary fragments immobilised onto the membrane. The conditions of hybridisation, including the temperature and salt con centration, are critical for this process to take place effectively. This is usually referred to as the stringency of the hybridisation and it is particular for each individual gene probe and for each sample of DNA. A series of washing steps with buffer is then carried out to remove any unbound probe and the membrane is developed, after which the precise location of the probe and its target may be visualised. It is also possible to analyse DNA from different species or organisms by blotting the DNA and then using a gene probe representing a protein or enzyme from one of the organisms. In this way it is possible to search for related genes in different species. This technique is generally termed zoo blotting.

Fig2. Southern blot apparatus.

The same basic process of nucleic acid blotting can be used to transfer RNA from gels onto similar membranes. This allows the identification of specific mRNA sequences of a defined length by hybridisation to a labelled gene probe and is known as Northern blotting (a homage to Southern blotting). It is possible with this technique to not only detect specific mRNA molecules, but it may also be used to quantify the relative amounts of the specific mRNA. It is usual to separate the mRNA transcripts by gel electrophoresis under denaturing conditions since this improves resolution and allows a more accurate estimation of the sizes of the transcripts. The format of the blotting may be altered from transfer from a gel to direct application to slots on a specific blotting apparatus containing the nylon membrane. This is termed slot or dot blot ting and provides a convenient means of measuring the abundance of specific mRNA transcripts without the need for gel electrophoresis; it does not, however, provide information regarding the size of the fragments.

Design and Production of Gene Probes

 The availability of a gene probe is essential in many molecular biology techniques. A gene probe is defined as a single-stranded piece of DNA complementary to a desired target DNA sequence that is labelled with, for instance, a fluorescent dye or radioactive label.

The information needed to produce a gene probe may come from many sources. In some cases it is possible to use related genes, that is from the same gene family, to gain information on the most useful DNA sequence to use as a probe. In addition, protein or DNA sequences from different species may also provide a starting point with which to produce a so-called heterologous gene probe. However, the increasing number of DNA sequences in databases such as Genbank and the availability of bio informatics resources has ensured that this is the usual starting point for gene probe design. Indeed, the method of choice of DNA production for probes is now almost exclusively by artificial chemical synthesis.

In this process, the chemical production of single-stranded DNA is undertaken by computer-controlled gene synthesiser machines. These link the monomers of DNA together based on the desired input sequence to form an oligonucleotide. However, the monomers used in chemical synthesis are so-called phosphoramidites , which are modified dNTPs ( deoxyribonucleoside triphosphates) that are joined via the 5′ carbon; this is in the reverse direction to natural DNA synthesis (carried out by DNA polymerase), where monomers are added to the 3′ end. Oligonucleotide primers used in the PCR are all produced by this method. This process allows alternative nucleotides to be used in the synthesis, which may be useful for optimising protein production and in altering protein properties by protein engineering.

Advances in the technology of chemically synthesising DNA has also led to the production of larger custom-made whole-gene sequences. Small genomes and chromosomes such as chromosome 3 from the yeast Saccharomyces cerevisiae have all been produced and this process has given rise to the new fi eld of synthetic biology, which is having a wide-ranging impact in biosciences and biotechnology.

Where little DNA information is available to prepare a gene probe, it is possible in some cases to use the knowledge gained from analysis of the corresponding protein. Thus it is possible to isolate and purify proteins and sequence part of the N-terminal end or an internal region of the protein. From our knowledge of the genetic code, it is possible to predict the various DNA sequences that could code for the protein, and then synthesise appropriate oligonucleotide sequences chemically. Due to the degeneracy of the genetic code, most amino acids are coded for by more than one codon, therefore there will be more than one possible nucleotide sequence that could code for a given polypeptide (Figure 3). The longer the polypeptide, the greater the number of possible oligonucleotides that must be synthesised. Fortunately, there is no need to synthesise a sequence longer than about 20 bases, since this should hybridise efficiently with any complementary sequences, and should be specific for one gene. Ideally, a section of the protein should be chosen that contains as many tryptophan and methionine residues as possible, since these have unique codons, and there will therefore be fewer possible base sequences that could code for that part of the protein. The synthetic oligonucleotides can then be used as probes in a number of molecular biology methods.

Fig3. Oligonucleotide probes. Note that only methionine and tryptophan have unique codons. It is impossible to predict which of the indicated codons for phenylalanine, proline and histidine will be present in the gene to be probed, so all possible combinations must be synthesised (16 in the example shown).

Labelling DNA Gene Probe Molecules

An essential feature of a gene probe is that it can be labelled and subsequently visualised by some means. This allows flagging or identification of any complementary sequence recognised by the probe.

The most common radioactive label is phosphorus-32 (³²P), although for certain techniques sulfur-35 (³⁵S) and tritium (³H) are used. These may be detected by the process of autoradiography where the labelled probe molecule, bound to sample DNA, located for example on a nylon membrane, is placed in contact with a film sensitive to X-rays. Following exposure, the film is developed and fixed, just as a black-and-white negative. The exposed film reveals the precise location of the labelled probe and therefore the DNA to which it has hybridised.

Non-radioactive labels are increasingly being used to label DNA gene probes. Until recently, radioactive labels were more sensitive than their non-radioactive counterparts. However, recent developments have led to similar sensitivities that, when combined with their improved safety, have led to greater acceptance.

The labelling systems are termed either direct or indirect. Direct labelling allows an enzyme reporter such as alkaline phosphatase to be coupled directly to the DNA. Although this may alter the characteristics of the DNA gene probe, it offers the advantage of rapid analysis, since no intermediate steps are needed. However indirect label ling is at present more popular. This relies on the incorporation of a nucleotide that has a label attached. At present, three commonly used labels are biotin, fluorescein and digoxygenin. These molecules are covalently linked to nucleotides using a car bon spacer arm of 7, 14 or 21 atoms. Specific binding proteins may then be used as a bridge between the nucleotide and a reporter protein such as an enzyme. For example, biotin incorporated into a DNA fragment is recognised with a very high affinity by the protein streptavidin. This may either be coupled or conjugated to a reporter enzyme molecule such as alkaline phosphatase, which converts the colourless substrate p -nitrophenol phosphate (PNPP) into a yellow-coloured compound p -nitrophenol (PNP) and also offers a means of signal amplification. Alternatively, labels such as digoxygenin incorporated into DNA sequences may be detected by monoclonal antibodies, again conjugated to reporter molecules such as alkaline phosphatase. Thus, the detection of non-radioactive labels occurs by either chemiluminescence or coupled reactions whose products allow for spectrophotometric detection. This has important practical implications since autoradiography may take 1–3 days, whereas colour and chemiluminescent reactions take minutes.

End-Labelling of DNA Molecules

The simplest form of labelling DNA is by 5′ or 3′ end-labelling. 5′ end-labelling involves a phosphate transfer or exchange reaction where the 5′-phosphate of the DNA to be used as the probe is removed and in its place a labelled phosphate, usually ³²P, is added. This is frequently carried out by using two enzymes. The first, alkaline phosphatase, is used to remove the existing phosphate group from the DNA. Subsequently, a second enzyme, polynucleotide kinase, is added that catalyses the transfer of a phosphate group (³²P -labelled) to the 5′ end of the DNA. The newly labelled probe is then purified by size-exclusion chromatography and may be used directly ( Figure 4).

Fig4. End-labelling of a gene probe at the 5′ end with alkaline phosphatase and polynucleotide kinase.

Using the other end of the DNA molecule, the 3′ end, is slightly less complex. Here, a new dNTP that is labelled (e.g. ³²P -αdATP or biotin-labelled dNTP) is added to the 3′ end of the DNA by the enzyme terminal transferase (Figure 5). Although this is a simpler reaction, a potential problem exists because a new nucleotide is added to the existing sequence and so the complete sequence of the DNA is altered, which may affect its hybridisation to its target sequence. A limitation of end-labelling methods arises from the fact that only one label is added to the DNA, so probes labelled in such a fashion are of a lower specific activity in comparison to probes that incorporate labels along the length of the DNA.

Fig5. End-labelling of a gene probe at the 3′ end using terminal transferase. Note that the addition of a labelled dNTP at the 3′ end alters the sequence of the gene probe.

Random Primer Labelling of DNA

The DNA to be labelled is first denatured and then placed under renaturing conditions in the presence of a mixture of many different random sequences of hexamers or hexanucleotides. These hexamers will, by chance, bind to the DNA sample wherever they encounter a complementary sequence, and so the DNA will rapidly acquire an approximately random sprinkling of hexanucleotides annealed to it. Each of the hexamers can act as a primer for the synthesis of a fresh strand of DNA catalysed by DNA polymerase since it has an exposed 3′-hydroxyl group. The Klenow fragment of DNA polymerase is used for random primer labelling because it lacks a 5′ to 3′ exonuclease activity. The Klenow fragment is prepared by cleavage of DNA polymerase with subtilisin, giving a large enzyme fragment that has no 5′ to 3′ exonuclease activity, but which still acts as a 5′ to 3′ polymerase. Thus, when the Klenow fragment is mixed with the annealed DNA sample in the presence of dNTPs, including at least one that is labelled, many short stretches of labelled DNA will be generated (Figure 6). In a similar way to random primer labelling, the PCR may also be used to incorporate radioactive or non-radioactive labels.

Fig6. Random primer gene probe labelling. Random primers are incorporated and used as a start point for Klenow DNA polymerase to synthesise a complementary strand of DNA, whilst incorporating a labelled dNTP at complementary sites.

A further traditional method of labelling DNA is by the process of nick translation. Low concentrations of DNase I are used to make occasional single-strand nicks in the double-stranded DNA that is intended as the gene probe. DNA polymerase then fills in the nicks, using an appropriate dNTP, at the same time making a new nick to the 3′ side of the previous one (Figure 7). In this way the nick is translated along the DNA. If labelled dNTPs are added to the reaction mixture, they will be used to fill in the nicks, and so the DNA can be labelled to a very high specific activity.

Fig7. Nick translation. The removal of nucleotides and their subsequent replacement with labelled nucleotides by DNA polymerase I increase the label in the gene probe as nick translation proceeds.

Fluorescence-Based Probes

 Molecular beacon probes contain a fluorophore at one end of the probe and a quencher molecule at the other. The oligonucleotide has a stem–loop structure where the stems place the fluorophore and quencher in close proximity. The loop structure is designed to be complementary to the target sequence. When the stem–loop structure is formed, the fluorophore is quenched by fluorescence resonance energy transfer ( FRET), i.e. the energy is transferred from the fluorophore to the quencher and ultimately released in a non-radiative fashion. The elegance of these types of probe lies in the fact that upon hybridisation to a target sequence, the stem and loop move apart, the quenching is then lost and emission of light occurs from the fluorophore upon excitation. These types of probe are used to detect DNA amplification accomplished by the polymerase chain reaction (PCR; see next section) and have the advantage that unhybridised probes need not be removed, therefore allowing uninterrupted monitoring of the reaction.

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مواضيع ذات صلة

Genes and Genome Complexity

Structure of Nucleic Acids

Genomics: Genome-Wide Analysis of Gene Structure and Function

Chromosomal Organization of Genes and Noncoding DNA

RNA Interference Causes Gene Inactivation by Destroying the Corresponding mRNA

Linkage Studies Can Map Disease Genes with a Resolution of About 1 Centimorgan

DNA Polymorphisms Are Used as Markers for Linkage Mapping of Human Mutations

The Two- Hit Theory of Tumor Suppressor Gene Inactivation in Cancer

DNA Microarrays Can Be Used to Evaluate the Expression of Many Genes at One Time

Hybridization Techniques Permit Detection of Specific DNA Fragments and mRNAs

Cloned DNA Molecules Can Be Sequenced Rapidly by Methods Based on PCR

Cellular and Molecular Mechanisms in Development: Gene Regulation by Transcription Factors