Alternatively, the protein products of equivalent donor and recipient genes can often be distinguished on the basis of size, charge, or enzymatic activity. By using an appropriate assay on cell extracts from hybrid cells, the presence of donor enzyme activity can be established. Another method is available for genes that encode proteins present on the cell surface. Antibodies raised against these proteins can be used to identify and separate hybrid cells that express such genes from those that do not. This process has been simplified by the use of antibodies with fluorescent labels and a fluorescence-activated cell sorter.

Finally, the presence of any DNA sequence can be detected in hybrid cells by nucleic acid hybridization. Under appropriate conditions, sequence-specific annealing of two complementary single-stranded DNA molecules occurs. A sequence is tested for by the isolation and immobilization of genomic DNA from the hybrid cell on a nylon membrane. Incubation of the membrane with a radioactively labeled single-stranded DNA fragment (probe) in aqueous solution allows the formation of double-stranded molecules between the probe and complementary hybrid DNA sequences. Such duplexes can be detected by autoradiography with x-ray film. Nucleic acid hybridization permits the mapping of any DNA sequence in hybrid cells, including polymorphic DNA markers, independent of the expression of a human gene in hybrid cells.

Isolated DNA sequences can also be mapped by in situ hybridization in which radiolabeled DNA probes are directly hybridized to whole chromosome preparations. The location of the hybridized probe sequence is shown by the presence of exposed film grains over the chromosome following autoradiography with photographic emulsion. Improvements in the preparation of chromosomes and image-enhanced microscopy permit the accurate mapping of DNA, sequences by this technique.

Linkage mapping
The order and distance between genetic loci on the same chromosome can be determined by the frequency of recombination events between them. Recombination between homologous chromosomes occurs during meiosis and results in the exchange of chromosomal regions.

The probability of a recombination event occurring between two loci is proportional to the distance between them. Thus, the frequency of recombination between two loci provides an estimate of the distance separating them. Positioning of a third locus can be achieved by determining the frequency of recombination which occurs between it and the other two loci. The stepwise analysis of many genetic loci in the manner can be used to generate a linkage map of each chromosome.

Accurate determination of linkage between two loci requires the analysis of large numbers of individuals from multigeneration families for recombination events. In human populations these are seldom available, and it is thus necessary to combine the information obtained families by using so-called lod (log odds) gives the probability that a particular combination of traits would have occurred linked with a particular recombination defined by the following equation

lod = log10 probability of linkage between two loci assuming σ = σ / probability of the two loci being unlinked (σ = 0.5)

Lod scores from individual families can cumulative score. A lod score of 3.0 is co for linkage, and one of -2.0 is indicative of its absence. The observation of recombination even the possibility of distinguishing between equivalent loci on homologous chromosomes. Only loci with a minimum of two alleles can be used in linkage analysis, and these alleles must be present in the population at a reasonable frequency to be informative. Although some genes, such as those for blood groups, have suitable allelic frequencies the number is limited.

Polymorphisms at the DNA sequence level, called restriction-fragment-length polymorphisms, have provided an unlimited source of codominantly inherited markers ideal for the construction of linkage maps. Restriction enzymes isolated from bacteria, recognize and hydrolyze DNA at specific 4-8 bp sequences. More than 200 restriction enzymes are known each of which recognizes a particular sequence.

Any sequence change within a restriction enzyme recognition site prevents cleavage there. In the absence of such a site, cleavage with the enzyme produces a larger DNA fragment. This absence can be detected by Southern blotting , in which genomic DNA is cleaved by the restriction enzyme and fractionated according to size by electrophoresis through a gel matrix. The DNA segment near the polymorphic site
Another useful type of polymorphism is provided by minisatellite DNAs, composed of repeated short DNA sequences. The number of repetitions at particular loci varies widely within the population, providing a set of highly
s
Fig. Southern blotting. (a) Genomic DNA is digested singly and doubly with restriction enzymes A, B, and C. (b) Digested DNA is fractionated by size-dependent migration through a gel matrix in an electric field. (c) Denatured DNA is transferred and immobilized onto a nylon membrane. (d) The filter is incubated with a radiolabeled single, stranded probe in aqueous solution. (e) Duplexes formed between the single-stranded probe and genomic DNA are detected by autoradiography, showing the calculated size of DNA fragments. (f) Comparison of the products obtained from single and double digestion allows the construction of a restriction map (1 kb = 1000 base pairs).

DNA sequence polymorphisms. (a) Restriction-fragment-length polymorphisms. A polymorphic restriction sit R* is detected by the probe. A 3-kb fragment is produced when the s' e is absent, a d a 2-kb fragment when it is present. (b) Variable number of tandem repeats. The number of repeat units changes the size of the fragment detected by digestion with the restriction enzyme R and he probe. The number of repeat units at a particular locus varies great between individuals.

(a)

(b)

polymorphic, multiallelic genetic markers ideal for linkage mapping. They can be used like restriction-fragment-length polymorphisms by cleaving the DNA with a restriction enzyme that does not have a site within the repeated sequence, followed by Southern blotting.
A linkage map constructed from restriction-fragment-length polymorphisms and variable-number-o -tandem-repeats loci can provide the starting point for gene mapping. By establishing linkage between a gene an known polymorphic markers, the position of that gene can be determined. That technique is particularly useful for mapping of genes without a known biochemical function, such s those involved in diseases. Furthermore, closely linked markers can be used for prenatal diagnosis in families with a hereditary disease. The cosegregation of a linked marker with a disease gene permits the prediction of whether an offspring has the disease gene simply by following the inheritance of the marker. Linkage analysis also permits the simultaneous mapping of multifactorial traits to many chromosomal regions.
Fine structures

Recombinant DNA technology has led to the mapping and analysis of genes at a molecular level. Fundamental to this has been the ability to produce large quantities of highly purified DNA sequences for further analysis by using molecular cloning techniques. Restriction enzymes can be used to insert DNA sequences into vectors propagated in bacterial hosts. Plasmid vectors consist of small, self-replicating circles of DNA, which can be used to clone segments of DNA of up to 10,000 bp (10 kilobases, or kb). Larger segments can be propagated in vectors derived from bacteriophage l. Part of the l. genome is not essential for lytic growth and can be replaced with up to 20 kb of exogenous DNA. This recombinant DNA molecule is incorporated into phage particles in the test tube and used in multiple rounds of infection of Escherichia coli cells. The DNA segments of 45 kb can be cloned into cosmid vector , which contain only the sequences from bacteriophage necessary for incorporation in phage particles. However, these can be used only for a single round of infection and are often unstable.

The first stage in determining the primary structure of a DNA molecule is the construction of a restriction map. The DNA is digested singly and with pairs of a set of restriction enzymes; the products of each digestion are subjected to gel electrophoresis. A comparison of the products obtained from single and double digests allows the order and spacing of restriction sites along the molecule to be determined. A restriction map provides the starting point for further manipulation and analysis of DNA molecules.

The order of nucleotides in small restriction fragments can be determined by DNA sequencing. In one method, a complementary sequence to a single-stranded template is synthesized by the enzyme DNA polymerase. Synthesis of the complementary strand is terminated at specific residues by the incorporation of a modified nucleotide. A separate reaction is performed for each nucleotide and results in a family of molecules, each of which terminates at that nucleotide. These products can be fractionated on the basis of size by electrophoresis through an acrylamide gel, and the sequence can be read from a ladder of fragments of different size.
The expression of a gene is dependent upon the synthesis of a messenger RNA (mRNA) molecule and its translation into the amino acid sequence of a protein by ribosomes in the cytoplasm. The DNA copies of mRNA (complementary DNA, or cDNA) from cells are synthesized by using the viral enzyme reverse transcriptase, and cloned for further analysis.

DNA sequencing. (a) A complementary strand of DNA is synthesized to a single-stranded template by the enzyme DNA polymerase. Chain growth is terminated upon the incorporation of a modified nucleotide, in this case A*, which results in a family of  fragments  each of the other three nucleotides, T, G, and C. (b) the products of each reaction are fractionated according to size by electrophoresis. The order of nucleotides is read from  the bottom of electrophoresis. The order of nucleotides is read from the bottom of electrophoresis. The order of nucleotides is read from the bottom of the gel (the smallest fragment) to the top.

Comparison of cDNA with genomic sequences reveals that the DNA encoding many eukaryotic genes is discontinuous. The blocks of coding sequences (exons) of a gene are brought together in the mRNA molecule by the splicing out of intervening sequences (introns). The presence of introns results in genes being distributed over a much larger stretch of DNA than would be expected from the analysis of their mRNA or protein sequences. The size and number of exons and intronsvary widely between genes. For instance, the b -globin gene contains only two introns of 14 and 331 bp and encodes a 620- bp mRNA. In comparison, the gene disrupted in Duchenne's muscular dystrophy encodes a 16-kb mRNA that comprises at least 60 exons and extends over 2 xl06 bp of DNA.

Mapping of the intron-exon structure of a gene-can initially be approached by using restriction enzymes. Restriction sites present in the genomic DNA but absent in the cDNA indicate the presence of some introns. Other techniques can provide higher resolution of the boundaries, but the intron-exon structure can be precisely mapped only by DNA sequence analysis; this often requires the isolation of large sections of genomic DNA by chromosome-walking techniques.

A library for this is constructed by partially digesting genomic DNA with a frequently cutting restriction enzyme. This produces a series of overlapping DNA fragments that cover the entire genome. These are cloned into bacteriophage or cosmid vectors, and the resulting library is screened by hybridization with a radioactive probe. Positively reacting clones that contain genomic DNA surrounding the probe are purified and restriction-mapped. New probes are isolated from the ends of these clones and used to obtain a second set of clones that overlap the original clone. Repetition of this process results in the isolation of a series of clones that contain an ordered set of overlapping genomic fragments.

The isolation of genomic sequences surrounding a gene by chromosome walking may result in- the mapping of gene clusters. For example, the human b-globin gene family stretches over 50 kb on chromosome 11. It contains five related functional genes that are active at different stages in human development. All of the genes are composed of three exons and probably arose from a single ancestral gene. Duplication of the ancestral gene by unequal crossing-over followed by sequence divergence over time has resulted in the evolution of five related but distinct genes. A homologous but functionally inactive b-globin pseudogene was also mapped within this region. Pseudogenes are genes that were presumably once active but have become defunct from the accumulation of mutations. The comparison of sequences surrounding genes has led to the identification of sequences involved in the regulation of genes.

Large DNA fragments
The resolution of linkage, analysis and chromosome in-situ hybridization only permits mapping to within a few million base pairs of DNA. That has stimulated the development of techniques for the manipulation and mapping of very large sections of DNA. Very large DNA fragments have been fractionated by using pulsed-field gel electrophoresis (PFGE). Whereas conventional gel electrophoresis can separate only fragments of less than 25 kb, pulsed-field gel electrophoresis resolves whole chromosomes of lower eukaryotes and DNA fragments of many thousands of kilobases. Long-range restriction maps can be constructed like conventional restriction maps by using pulsed-field electrophoresis in conjunction with restriction enzymes that cut infrequently in the human genome. These enzymes typically contain at least one rare CpG dinucleotide and are usually sensitive to its methylation. Although this dinucleotide is usually methylated, many genes are preceded by a short stretch of unmethylated DNA containing an abundance of CpG's. Thus the clustering of sites for a number of rare cutting restriction enzymes can be used with pulsed-field gel electrophoresis to predict where potential genes may lie.

Cloning of DNA stretches greater than 200 kb by chromosome walking is technically difficult. Isolation of DNA sequences a substantial distance away from an initial point may be achieved by using chromosome-jumping techniques. The basic for chromosome jumping involves the circularization of very large DNA fragments generated by digestion with infrequently cutting enzymes or partial digestion. This results in DNA regions that were far apart in the genome being brought together at the junctions of these circles. These junctions are selectively cloned into standard vectors. Clones isolated from such a jumping library contain DNA from two different loci, one reacting with the probe sequence and the other originating at a distance from it. A probe isolated from the second locus is used in the next jump. Each jump is monitored by using Southern blots of pulsed-field gels.

Very large DNA fragments can be cloned at1d propagated in yeast cells by constructing yeast artificial chromosomes (YACs). The ligation of a functional yeast centromere and telomere to the ends of a large DNA fragment results in the maintenance of this fragment chromosome in yeast cells. The technique could allow the cloning of DNA fragments of 500 kb and the analysis of large genes and gene families within a single clone. Furthermore, the entire human genome could be represented by 10,000 such clones. Determination of the linear order of these clones would permit the accurate mapping of loci by a simple screening procedure.

Molecular mutations
Many mutations are the result of small, subtle changes within the DNA sequence of a gene. The substitution of a single base pair within the reading frame of a gene may result in the incorporation of a different amino acid in the protein or premature termination of mRNA translation. These changes can eliminate or drastically alter the activity of a protein. For example, the reduced affinity of hemoglobin for oxygen in sickle cell anemia is caused by a single amino acid substitution resulting from a single base change in the b-globin gene. These mutations, as well as others, are mapped by comparing the DNA sequence of the mutant gene with that of the wild type. The sequence of specific regions of the human genome in large populations can be ascertained by the selective amplification of those regions by using the polymerase chain reaction. Oligonucleotides flanking the target sequence are used as primers for repetitive cycles of DNA synthesis of that sequence in the test tube (Fig. 4). The resulting amplified product can be directly subjected to DNA sequence analysis. Alternatively, the presence or absence of a known polymorphic restriction site within the product can be tested directly by enzyme digestion and gel electrophoresis. This simplifies the typing of individuals for restriction-fragment-length polymorphisms in linkage analysis. Furthermore, the magnitude of DNA amplification by polymerase chain reaction permits the analysis of single sperm cells. Each sperm cell is the product of a single meiosis. The mount of recombination occurring between two polymorphic loci can be accurately measured by simultaneously amplifying and typing those loci in a large number of individual sperm cells. Although this analysis is restricted to DNA sequence polymorphisms, it may provide a much simpler and more accurate method of measuring the genetic distance separating such loci than is offered by extensive family studies.

Fig. Polymerase chain reaction. Regions of DNA defined by flanking oligonucleotides are amplified by successive cycles of denaturation primer annealing, and chain elongation. Ideally, each round of amplification would double the target sequence. The resulting can be analyzed with restriction enzymes and by DNA sequencing.