Methods for finding genes a major rate-limiting step in positional cloning

29 GATA 10(2): 29-41, 1993

ORIGINAL ARTICLES

Methods for Finding Genes A Major Rate-Limiting Step in Positional Cloning JULIA E. PARRISH and DAVID L. NELSON

Identification of transcribed sequences from within genomic regions has been a major rate-limiting step in the pursuit of genes involved in many human genetic diseases. Early efforts focused primarily on screening of cDNA libraries, identification of evolutionarily conserved sequences, and northern blot hybridization. In recent years, several innovative techniques for gene identification have been devised. These techniques expand the size of the genomic region capable of being scanned for genes, while also allowing detection of genes regardless of their expression patterns. This article reviews several new and older techniques and discusses the advantages and limitations of each.

Introduction Many human genetic diseases have been pursued by the linked marker, positional cloning approach. A significant rate-limiting step in these pursuits is the identification of expressed sequences within limited genomic regions. Methods that enable the identification and isolation of expressed sequences from specific chromosomal regions and cloned sequences are quite valuable for the isolation of candidate disease genes. Furthermore, the ability to apply these methods to larger regions (such as cytogenetic bands or chromosome arms) would enhance the capacity to test for candidate genes at an earlier stage of the search, when linked markers are still some distance away, and at the same time enable a more complete description of such large regions with regard to the resident genes. This methodology will become increasingly important as the physical maps of human chromosomes are completed.

From the HumanGenomeCenter and Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas, USA. Address correspondence to Dr. D.L. Nelson, HumanGenome Center and Institute for Molecular Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. Received 23 April 1993; accepted 28 April 1993.

Methods for isolation of human genes based on their chromosomal location (positional cloning) [1, 2] were first devised in the late 1970s. Three developments spurred interest in this approach at that time. The first was the recognition that restriction fragment length polymorphisms (RFLPs) could be used to define and follow transmission of loci in families with inherited disorders [3, 4] and for construction of linkage maps in normal humans [5]. Initially, generation of RFLPs took a random approach, with no specific chromosome or region targeted. As specific chromosomes became interesting for study, emerging technology was used to enable isolation of DNA fragments from specific chromosomes. Flow-sorted chromosomes were used to establish libraries of small DNA fragments specifically derived from a single human chromosome. These libraries could be used to generate probes with potential polymorphic variation. Probes could be regionally assigned using either genetic or physical (somatic cell hybrid [6]) mapping methods. The recognition that human and rodent DNAs could be distinguished by their interspersed repetitive sequences [7] enabled isolation of human-specific clones from somatic cell hybrids retaining either intact or fragmented human chromosomes. The ability to target specific regions retained in hybrids provided greater resolution of this method over use of flowsorted libraries. While these three basic methods have evolved, they still form the basis for many of the techniques of positional cloning in use today. Modem genetic maps continue to use DNA variation; however, the short tandem repeat (STR or microsatellite) sequence is now the marker of choice [8]. Flow-sorted libraries have improved such that cosmid and yeast artificial chromosome (YAC) libraries have been constructed directly from flow-sorted chromosomes [9]. And somatic cell hybrids continue to be extremely useful reagents both for isolation of DNA to construct libraries [10, 1 1] and for production of probes using Alu PCR to identify cloned fragments from specific regions [12, 13]. Methods for identification and isolation of genes in a chromosome- or region-specific manner have lagged behind the methods described above for isolation of random genomic DNAs. One of the earliest attempts to isolate genes in a chromosome-specific manner was described in two articles in 1986, using specific detection of human sequences with repetitive elements. A cDNA library constructed from a somatic cell hybrid retaining chromosome 21 as its only human material was screened for clones containing the human Alu repetitive element [14, 15]. This attempt did yield three clones (from a library of

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30 J.E. Parrish and D.L. Nelson

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1,000,000) that contained Alu repeats. This early method was later modified using nuclear RNA [16]. The earliest method using anonymous cloned DNAs to identify genes employed the "zoo blot" approach and was instrumental for isolation of the dystrophin gene involved in X-linked Duchenne muscular dystrophy [17]. At present, a large number of methods building on and extending from these early experiments are at various stages of development. This article describes a number of these and points out the advantages and limitations of each. Substantial efforts are under way to assemble overlapping clone maps of large regions of human chromosomes by using YACs [18, 19]; untold numbers of smaller projects involve assembly of such contigs within specific portions of the genome that are suspected to contain genes of medical or biological interest. Since YAC contigs provide such an important resource for cloning large regions of the genome in a small number of clones (see Schlessinger and Kere [20] for a review of YAC-based physical mapping), the development of efficient methods for locating genes contained in YAC clones is an essential component in the success of numerous projects. Much effort has been focused on this area of research, and several different methods of locating coding sequences within YAC clones have been devised. In some cases, a search for genes can be focused on a region of interest for which no cloned DNAs are available. Several methods for direct isolation of cDNAs from such regions have been devised. These cDNA clones, in turn, can be used to identify YAC clones, thus leading directly to a physical map around genes within the region of interest.

Methods Using Cloned DNA Expression-Dependent Methods Many methods are available for isolating cDNA clones directly from cDNA libraries by using cloned probes. Cloned DNA fragments may also be used as probes against Northern blots in order to assess the possibility of expressed sequences being found within a given fragment. All of these methods have the universal disadvantage that, in order to be detected, the gene in question must be expressed in the tissue from which the cDNA library was made.

Screening cDNA Libraries with Complex Probes. YAC inserts can be directly labeled and used as probes for screening cDNA libraries [21]. This method has the advantage of obviating subcloning of the YAC,

and multiple transcripts can be identified with a single screening. There are significant disadvantages to this approach, however. First, it is necessary to isolate YAC DNA in sufficient quantity and purity for labeling, which is a significant effort in itself (see Nelson [22] for review of methods). Second, long incubation times (16 h or more) are required for sufficient incorporation of isotope in labeling reactions. Third, the probe itself is complex and contains large numbers of repetitive sequences, leading to poor signals, increased background, and false positives. Fourth, clones containing short exons or poorly represented cDNAs might be missed entirely, although the use of normalized cDNA libraries [23] can circumvent the latter difficulty. Lovett et al. [24] conducted a similar screening of a cDNA library that had been enriched for YAC sequences by hybridization of the library to immobilized YAC DNA (see below). This screening resulted in positive identification of only three of five known positive clones.

Northern Blot Analysis. Screening genomic clones for hybridization to RNA in Northern blots shares some of the advantages and disadvantages of direct screening of cDNA libraries. The transcript must be present in the tissue from which RNA was derived, and in sufficient abundance to be detected. YAC inserts are too complex to reliably identify transcripts on Northern blots; however, cosmids and phage have successfully detected transcripts [25], so subcloning of YACs into cosmid or phage contigs is indicated in this case. Subclones that detect transcripts can then be used to screen cDNA libraries. This enables a downsizing of the DNA used as probe in screening, a significant advantage over using YACs directly as cDNA library screening probes. However, the process of subcloning the YAC and individually testing each subclone for hybridization to Northern blots makes this method quite tedious, especially when large genomic regions are to be analyzed. Hybrid Selection. Recently, three "hybrid selection" techniques have been devised that circumvent many of the disadvantages of the above methods. All three methods employ the polymerase chain reaction (PCR) [26] in conjunction with selection of cDNAs by hybridization to large genomic inserts. The first strategy [27] entails the immobilization of target DNA (purified YAC or cosmid DNA) on nylon-filter discs (Figure 1A). Repeated sequences are quenched by prehybridization of the target DNA on filter discs with a mixture of DNAs from a chromosome 15 genomic library, a genomic repetitive

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31 GATA 10(2): 29-41, 1993

Gene Identification

sequence library, rRNA-specific clones, poly(dI)poly(dC), and yeast DNA. Following this step, a total cDNA library, prepared using random hexamer primers, is allowed to hybridize to the target DNA. Nonspecifically hybridizing sequences are removed, and the specific cDNAs that remain are amplified by PCR using a series of nested primers directed to cDNA library vector sequences. This technique produced a 7000-fold enrichment in the abundance of a rare transcript after two rounds of selection in control experiments. The second strategy [24] differs substantially from the first in two ways. First, repeated sequences are quenched by hybridization in solution of the cDNA library with sheared total human DNA, with pBR322 and yeast DNA included in some cases (Figure 1A). Second, only one pair of primers is used for amplification. This technique produces levels of enrichment similar to the former (800- to 2000-fold in one cycle of selection). The third strategy [28] differs in that hybridization is carried out in solution. PCR-amplified cDNAs are hybridized to biotinylated cosmid DNA; streptavidincoated magnetic beads are then used to immobilize

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the target DNA for removal of nonspecific cDNAs, followed by elution and amplification of specific cDNAs (Figure 1B). These methods for cDNA selection offer several advantages over direct screening methods. They can be rapidly accomplished and are insensitive to the presence of introns or cryptic splice sites, which can cause problems with the exon-trapping schemes outlined below. They enable detection of rare transcripts (<1 part in a few million [28]). They can detect human genes that have diverged from those of other species, since cross-species homology is not required in the selection scheme. The presence or absence of CpG islands [29] does not affect the selection process. These methods are virtually insensitive to the size of the genomic target fragment; similar levels of enrichment have been obtained with cosmids and YACs, indicating that YACs need not be subcloned. The potential exists for a single gel and blot containing several different YAC clones to select cDNAs for each; this represents an enormous improvement over methods that require isolation of YAC DNA from each clone, followed by individual labeling and screening.

wash; elute specific cDNAs

,: I

Figure 1. Diagram of hybrid selection schemes. (A) In the scheme described in Parimoo et al. [27] (a), repeated sequences are quenched by prehybridization of the genomic DNA on filter discs with blocking DNAs. The filters are then hybridized with the cDNA library, nonspecific cDNAs are removed, and specific cDNAs are eluted and amplified. In the scheme described in Lovett et al. [24] (b), repeats are quenched by prehybridization of the cDNAs with blocking DNA. (!1) In the scheme described in Korn et al. [28], target genomic DNA (cosmid) is biotinylated and prehybridized with blocking DNA. After hybridization with cDNA, the biotinylated DNA is immobilized by complexing to streptavidin-coated magnetic beads. Nonspecific cDNAs are removed, and specific cDNAs are eluted and amplified. © 1993 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010

32

J.E. Parrish and D.L. Nelson

GATA 10(2): 29-41, 1993

As mentioned above, an inherent disadvantage to any method employing a cDNA library is that the proper library (one containing transcripts from the region of interest) is vital to the success of the selection. This problem can be avoided through the use of pooled cDNA libraries, and the selective power of these methods suggests rare transcripts can be found in quite complex mixtures. Yeast contamination in cDNA libraries can lead to selection artifacts, particularly when total yeast DNA is used as the target DNA, rather than the isolated YAC. Finally, to identify positive clones unequivocally, it is necessary either to hybridize individual cDNA clones to Southern blots of YAC or cosmid clones [27] or to screen enriched libraries with labeled YAC DNA directly [24]. In the latter case, the enrichment of the library increases the likelihood of obtaining positive signals, but the screening itself remains problematic in terms of low signal and high background.

Expression-Independent Methods Methods that do not require expression of a gene in the initial phases of identification provide distinct advantages over the methods discussed above. Primarily, they remove the dependence upon having a cDNA library made from the appropriate tissue at the appropriate developmental time. Once a potential coding sequence is identified by these methods, expression must still be confirmed either through Northern blot hybridization or by isolation of a cDNA; however, these stages are approached with a smaller fragment that is already suspected to contain potential exons.

Cross-Species Sequence Homology. Cross-species sequence homology [17, 30-33] has been quite successful in identifying potential coding sequences. Based on the observation that coding sequences are much more highly conserved between species than are noncoding sequences, this analysis involves hybridization of a candidate DNA fragment to DNAs from a variety of species to determine whether the candidate cross-hybridizes across species (or class, order, or phylum) boundaries. A clone with hybridizing correlates in other species is generally found to contain coding sequences. Since this method utilizes genomic DNA rather than RNA, it is more dependable than Northern blot hybridization. This method does have some distinct disadvantages as a general method for locating all genes in a region. First, subcloning of large DNA fragments is necessary, a significant problem when very large regions

are to be searched. Since each subclone must then be individually hybridized to the "zoo blots," this can represent an enormous effort. Second, genes that have diverged significantly from other species will not be detected.

Selection for CpG Islands. In recent years, the technique of scanning a region for CpG islands [29] has become commonly used. Since small, unmethylated CpG-rich regions are frequently associated with expressed sequences, and since these "islands" can be detected by cleavage with rare-cutting restriction enzymes, this technique provides a rapid means for assaying a large stretch of DNA in a relatively short period of time. CpG islands are small regions of genomic DNA containing cleavable sites for several different rare-cutters; while cloned DNAs may contain many such clusters, not all of these will be present in genomic DNA due to differences in methylation. Therefore a cluster of rare-cutter sites found in a cloned DNA fragment must be shown to be cleavable in genomic DNA before it can be considered a CpG island, although, in practice, most clusters of rare-cut sites found in cloned DNA represent true islands. Clones that lie near CpG islands can be used as probes against Northern blots or cDNA libraries. This approach has led to successful isolation of genes in numerous instances [34-39]. It carries the additional advantage of enabling detection of lessconserved genes than might be detected by hybridization to zoo blots. The only significant disadvantages are that the region in question must be analyzed by pulsed-field gel electrophoresis for the presence of sites for rarely cutting restriction enzymes, and that DNA fragments near these sites must be cloned. A novel method for circumventing these difficulties, which was described by Patel et al. [40], involves digestion with Not I, ligation of an adaptor, and PCR between an adaptor primer and an Alu primer (Figure 2). PCR products are then cloned and analyzed. The original report described the application of this technique to radiation-reduced hybrid cell lines, each of which contained a small amount of human DNA. A recent report by Valdes et al. [41] described the application of this technique to YACs. An important drawback to this method is that not all genes are associated with CpG islands; for those that are, the clones that map near the CpG island still must be further analyzed in order to determine whether they represent transcribed sequences and, if so, to isolate cDNAs representing these genes. Exon Trapping. Innovative techniques have been

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33 Gene Identification

GATA 10(2): 29-41, 1993

N° I ,

Figure 2. Selection for CpG islands. Genomic DNA is digested with rare-cutting restriction enzymes (Not I in this case), linkers are ligated onto the ends, and PCR is carried out using a linker-specific primer and an Aluspecific primer. When an exon is flanked by a CpG island and an Alu repeat, it will be amplified and can then be used to probe cDNA libraries.

exon

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devised that enable identification of exons directly from genomic clones. The first such exon-trapping scheme was devised by Duyk et al [42]. The strategy involves shotgun cloning genomic DNA into a trapping vector that contains a splice donor site. Further manipulations, including transfection into mammalian cells, then enable clones that contain a splice acceptor site to be identified. This system requires multiple cloning steps and is quite laborious. The exon-trapping scheme devised by Buckler et al. [43] is much simpler to use and requires the presence of both splice acceptor and splice donor sites in the insert DNA (Figure 3). This vector can accommodate relatively large DNA fragments and can be used to test many fragments in a "shotgun" fashion, thus simplifying the rapid analysis of larger regions of the genome. Exon-trapping schemes are theoretically able to detect all of the internal exons in a given region of interest, regardless of their states of transcriptional activity. The introduction of vectors requiring both splice acceptors and donors [43] provides an increase in the specificity and sensitivity of the system by reducing the likelihood of detecting cryptic splice sites. This method has been applied to several regions recently with excellent results (see Table 1).

Screeningfor Splice Sites. Melmer and Buchwald [44] describe a method for screening for genes by using oligonucleotides corresponding to splice site consensus sequences as hybridization probes. Random phage and cosmid DNAs were digested and transferred to membranes. Degenerate oligonucleotides were designed: a 4096-fold degenerate 15-mer and a 256-fold degenerate 9-mer directed at the 3' splice site, and a 128-fold degenerate 10-mer directed at the 5' splice site. The 9-mer was expected to detect

--40% of all 3' slice sites, whereas the 10-mer was expected to detect - 1 8 % of all 5' splice sites. These oligonucleotides were labeled and hybridized to the membranes, and they detected bands in one-quarter to one-third of the clones tested. Ten cosmid clones containing hybridizing bands were tested for crossspecies homology, and all ten showed significant homology. Several positive phage clones were also hybridized to Northern blots. Signals were poor, but this is probably due to use of whole phage DNA as probe, rather than the isolated fragment responsible for the positive signal with the oligonucleotide. This method has the advantage of simplicity: an entire collection of clones (phage or cosmid) can be screened for the presence of splice sites in a single experiment. Although the probes cannot detect all splice sites, use of 5' and 3' oligonucleotides together increases the likelihood that at least one splice site in a given gene will be detected. Subcloning of positive fragments would facilitate further analysis, including Northern blot analysis, sequencing, and screening cDNA libraries.

Sequence-Based Methods Recent computational advances have enabled the development of sequence-based methods for identifying potential exons in genomic sequence data. Comprehensive databases allow for sequence comparison both between and within species; sequence conservation may indicate the presence of an exon. Ideally, analytical methods should not depend on a gene from another species having already been isolated and sequenced. Toward that end, an approach has been developed that uses neural net recognition to localize and characterize genes and other biologically important features in DNA sequence data [45]. The

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34 J.E. Parrish and D.L. Nelson

GATA 10(2): 29-41, 1993

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Table !. Human Disease Genes Isolated by Positional Cloning and Method of Identification Disorder

Year

Chrom

Chronic granulomatous disease Duchenne muscular dystrophy Retinoblastoma Cystic fibrosis Wilm's tumor Neurofibromatosis type 1 Testis determining factor Choroideremia Fragile-X syndrome Adenomatous polyposis coli

1986 1986 1986 1989 1990 1990 1990 1990 1991 1991

X X 13 7 11 17 Y X X 5

Aniridia Kallmann syndrome

1991 1991

11 X

Myotonic dystrophy Norrie disease Lowe syndrome Menkes disease X-linked agammaglobulinemia Glycerol kinase deficiency Adrenoleukodystrophy Neurofibromatosis type 2 Huntington disease

1992 1992 1992 1993 1993 1993 1993 1993 1993

19 X X X X X X 22 4

Method

Reference

Enriched cDNA screening of phage Zoo blot followed by cDNA screening Zoo blot followed by cDNA screening Zoo blot followed by cDNA screening Zoo blot followed by cDNA screening Zoo blot followed by cDNA screening "Noah's Ark" blot; sequence analysis Zoo blot followed by cDNA screening Direct cDNA screening Direct cDNA screening, SSCP analysis; zoo blot, sequence analysis, and cDNA screening Zoo blot, CpG island, and cDNA screening Direct sequence analysis and direct cDNA screening Triplet repeat, direct cDNA Direct cDNA screening Direct cDNA screening Exon trapping and direct cDNA screening Several Est and exon amplification Zoo blot, CpG island, and GRAIL Exon amplification Exon amplification and triplet repeat

60 17 61 25, 62, 63 64, 65 66 67 68, 69 70 71-73

GRAIL, gene recognitionand analysis internet link.

© 1993 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010

74, 75 76, 77 78-83 84, 85 86, 87 88-90 92 93, 94 91 95 96

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Gene Identification

GATA 10(2): 29-41, 1993

foundation of this project is a unique set of tools developed for recognizing the variable sequence patterns characteristic of gene sequence features. Each of these tools consists of two parts: (a) a group of algorithms, or sensors, to measure sequence attributes related to the feature of interest; and (b) a neural network that learns to recognize the feature by examining output from the sensor algorithms. This coding recognition module (CRM) has been combined with a rule-based interpreter and user interface to produce a gene recognition and analysis internet link (GRAIL), which enables users to submit DNA sequences by e-mail and electronically receive for each potential exon (a) an analysis of potential exon positions with strand assignment and preferred reading frame determination, (b) a qualitative evaluation, and (c) a protein database comparative search. Very short exons may be missed due to the 99-base sequence window used by the system; however, the system appears to be able to detect 90% of exons present in a given sequence, with - 2 0 % false positives. At least three other similar methods are currently in use [46--48]. One drawback to searching for genes in this manner is that, when a large region is to be searched, huge amounts of sequence data must first be generated. Alternative methods for identifying likely coding regions (that is, by the methods described above) could be used to narrow the scope of the sequencing project; computational systems could then be used to detect potential exons within these sequences.

Cloned DNA-Independent

Methods

Subtractive cDNA Hybridization Many subtractive hybridization techniques exist. Jones et al. [49] have developed an approach for the application of subtractive hybridization directly to cDNAs in order to isolate sequences that are expressed in one cell type but not in another (for example, two hybrid cell lines retaining different complements of human DNA). This application requires that one hybrid cell line retain a region of interest, while another hybrid cell line contains the entire complement of human DNA that is present in the first, except the region of interest. A hybrid retaining a deletion chromosome could be subtracted with a hybrid retaining only the intact chromosome in order to isolate transcripts specifically from the deletion region. The technique itself is relatively straightforward (Figure 4). Single-stranded, radiolabeled cDNA is synthesized from the cell line retaining the region of

interest. These fragments are then hybridized in solution with excess biotinylated poly(A) + RNA from the second cell line. Any cDNA from a region contained in both cell lines will have a corresponding biotinylated RNA and will form a heteroduplex. These biotinylated heteroduplexes are removed from the solution by binding with streptavidin and phenol extraction. After a second round of subtraction, the remaining labeled cDNAs are used directly to screen a cDNA library. The initial experiments with this system detected nine cDNAs from a 2- to 4-megabase region including the TSE 1 gene [49]; thus, the system appears to be quite sensitive in detecting transcribed genes. Genes that are not transcriptionally active in the particular cell line in question would not be detected. The most significant advantage to this method is its applicability to uncloned regions of DNA. This will be particularly useful for locating genes in regions of the genome where deletions are associated with phenotypic disorders. This would lead directly to potential disease genes, while at the same time providing clones from within the deleted region for the initiation of physical mapping and cloning of the region. An extension of this application could provide a simplified method for recovering transcripts from YACs. A YAC or contig could be transfected into mammalian cells, and these cells could then be subtracted with the parent cell line.

Coincident Sequence Cloning A unique PCR-based approach has been described [50] that enables recovery of common sequences from two partially overlapping DNA sources. When one of these sources is a cDNA library, the technique enables direct isolation of cDNAs specific for a region of human DNA contained within a hybrid cell line. The method is also applicable to cloned DNAs, including YACs. In the experiments described [50], human DNA and DNA from a hybrid cell line retaining human chromosome 11 and part of Xpter were used as the two DNA sources, and the technique identified seven unique human clones that were also present in DNA from the hybrid cell line. The procedure itself is relatively straightforward (Figure 5). DNA from one source is doubly digested and cloned into M13 to generate single-stranded DNA with defined ends. "Capture" oligonucleotides are annealed to the resulting mixture of DNAs. The other DNA source is digested with the same two enzymes and alkali denatured. The two mixtures of DNA are then allowed to anneal. Only fragments that are present in both DNA sources will anneal properly to form a

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36 J.E. Parrish and D.L. Nelson

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hybrid cell line B

hybrid cell line A

I

region of interest

D single-stranded, labeled cDNA

poly(A)+, biotinylated RNA

solution

Figure 4. The subtractive cDNA hybridization scheme of Jones et al. [49]. The heavy lines represent the portions of a human chromosome present in each hybrid cell line. The boxes represent cDNA and RNA produced from each hybrid. The most important consideration is that hybrid B must contain the entire complement of human DNA present in hybrid A except for the region of interest. Hybrid B may contain additional human DNA without affecting the procedure.

remove biotinylated RNA-cDNA by complexing with streptavidin and phenol extraction

use uncomplexed single-stranded cDNA for a second round of subtraction, then use directly to probe cDNA library

B

double-stranded fragment with capture oligonucleotides at each end. These fragments are then amplified by PCR using primers derived from the capture oligo sequences, and libraries are made from the products. This technique should prove useful in isolating genes from complex DNA sources, such as radiation-reduced hybrid cell lines or YAC clones, although with YAC clones the presence of contaminating yeast DNA in many cDNA libraries may present a problem. It could also be used to produce cDNA clones at random from a single human chromosome by using genomic DNA from a hybrid cell line retaining only that chromosome and a total human cDNA library.

Hybrid Cell/hnRNA Approaches Three methods have been described that take advantage of hnRNA transcripts from human genes in somatic cell hybrids. The first [16] uses degenerate hexameric oligonucleotides directed to consensus

splice donor sequences to prime cDNA synthesis from hnRNA. This targets unspliced RNAs and primes cDNA synthesis into the exon located immediately 5' of the primer-binding sequence. A large cDNA library was constructed and screened with total human DNA to identify clones derived from human transcripts. A number of clones were isolated and demonstrated to be derived from the human component of the hybrid. The second method [51] is similar to the first, except that random hexamers are used to prime cDNA synthesis. Like the first, this method requires construction of a large cDNA library and screening for human clones. Using this technique, Ellison et al. [51 ] were able to isolate three previously unidentified genes that are transcribed from an inactive X chromosome. The third method [52] relies on the presence of human Alu repetitive sequences in introns and 3' untranslated regions of human genes. This method takes advantage of oligonucleotides directed to Alu

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37 GATA 10(2): 29-41, 1993

Gene Identification

A

B

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I

Double digestion convert to single-stranded DNA with defined ends

Double digestion denature

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Figure 5. Coincident sequence cloning. Restriction enzymes 1 and 2 represent any pair of restriction enzymes. Only DNA that is present in both sources will form a heteroduplex that will amplify by PCR. Adapted from Brookes and Porteous [50].

l

Anneal

noncoincident DNA from A

Heteroduplex coincident DNA

noncoincident DNA from B

Ligate. Isolate and PCR amplify heteroduplex

( sequences for the construction of cDNAs. Since the majority of clones produced should be of human origin, construction and screening of a large library are unnecessary. The application of these primers to synthesize cDNA from human hnRNA present in a hybrid cell line retaining Xq24-qter enabled the isolation of a library of 100 cloned products, 80% of which localize to Xq24-qter. These methods should enable isolation of a significant fraction of the genes in a given region that are expressed in hybrid cell lines.

Microdissection/Microcloning A distinct disadvantage of the above methods for isolating genes from uncloned chromosomal regions is that each requires the production of hybrid cell lines that retain very specific portions of the genome. Such cell lines are somewhat laborious to produce,

•

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sites for restriction enzyme 1

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sites for restriction enzyme 2

and in some cases it may not be possible to obtain an appropriate cell line for a given purpose. Microdissection and microcloning offers one method that can be accomplished regardless of the availability of hybrid cell lines or clones. The only requirement is a knowledge of the chromosomal region in which the gene or genes of interest lie, and the necessary equipment and expertise to perform the microdissection and subsequent micromanipulations. In the method described by Ltidecke et al. [53], microdissection is performed on banded and stained chromosome preparations, the resulting DNA is ligated to vector sequences, enabling insert amplification by PCR, and the products are then cloned into a plasmid vector. This method can produce thousands of genomic clones from the region of interest. More recent innovations now enable amplification of microdissection products with no microchemical manipulations [54]. The dissected chromosome frag-

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38 J.E. Parrish and D.L. Nelson

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ment is added to a collecting drop containing proteinase K. After digestion, the proteinase K is heat inactivated, PCR reagents (including a universal primer) are added, and products are amplified. This technique represents a significant simplification over previously described methods [53]. The application of this technique to the isolation of coding sequences can be somewhat convoluted. The microclones can be used to seed YAC, phage, or cosmid contigs, and these can be searched for genes by one of the above methods. Alternatively, the microclones themselves can be used to screen cDNA libraries directly. In one case [55], two cDNA clones were isolated by using a total of 200 microclones. Even using pooled microclones, this represents a low rate of return for a significant effort. Considering the small size of the microclones produced by this procedure (170-1300 bp in Yu et al. [55]), it is unlikely that microcloning followed by direct screening will be generally applicable as a technique for isolating region-specific transcripts; however, using microclones as a hybridization substrate in cDNA capture schemes may also prove valuable.

Whole Genome Efforts Recently, massive efforts have been undertaken to partially sequence random cDNA clones from human brain cDNA libraries [56-59]. These sequences have been termed expressed sequence tags (ESTs), as they define gene products much like sequence-tagged sites (STSs) define genomic loci [56]. To date, >20,000 previously unidentified genes have been isolated. Currently, efforts are in progress to determine chromosomal localizations for these ESTs; a random distribution would provide, on average, >100 new markers per chromosome. ESTs can be used in the same manner as STSs for physical mapping purposes as well as for isolation and characterization of the genes from which they were derived. An EST that maps to a region of interest can be used both as a seed for a physical map of the region and as a candidate gene for genetic disorders in the vicinity. Such random approaches are outside the scope of small projects in which genes from a specific region are being sought, but the availability of these chromosomally localized cDNA clones to individual investigators will provide a tremendous service, while at the same time increasing the pace of the human genome project. As more ESTs are localized to each chromosome, it will become increasingly efficient to use a candidate gene approach to cloning disease

genes. It is likely that this approach will replace positional cloning in most instances.

Conclusions The techniques reviewed in this article have all proven useful in assisting efforts to clone genes from within defined portions of the human genome. The classic techniques (direct screening of cDNA libraries, crossspecies homology, and so on) have been successful in numerous positional cloning projects (see Table 1). The newly developed techniques are beginning to be more widely used and are enabling more rapid analysis of larger regions of the genome. For example, Morgan et al. [97] recently isolated nine unique cDNAs from a 425-kb YAC containing the human interleukin-4 and -5 genes by using a modified direct selection approach [24, 27]. Vetrie et al. [92], using a similar approach, have cloned the gene involved in X-linked agammaglobulinemia. As approaches to cDNA selection using YACs are refined and become more widely used, many more successful gene isolations are likely to take place. Given the variety of different techniques currently available for use in searching for genes from a given region of the genome, it seems likely that any positional cloning effort will succeed, given sufficient time. This conclusion is validated by the recent cloning of the gene responsible for Huntington's disease [96] after 10 years and an enormous amount of effort. Most efforts will probably entail the use of several different methods, in parallel or in series. A case in point is the characterization of the human major histocompatibility complex (MHC), in which many different methods were used to identify large numbers of genes in this very gene-rich region (for a review, see Milner and Campbell [98]). In cases where only small regions need to be searched, direct screening for cDNAs may function well; exon trapping could also be employed if no clones are detected. To scan larger regions thoroughly, painstaking and tedious procedures are still required. As the genome project proceeds, new and faster methods will undoubtedly become available, as the development of such methods has become a major focus in many laboratories. Of particular value would be methods for reliably locating all genes present on a given YAC, with no dependence on expression and no sequence data. Such methods would greatly accelerate the pace of most positional cloning efforts and would provide a muchneeded technological advance.

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J.E.P. is supported by the human genome distinguished postdoctoral fellowship program from the Office of Health and Environmental Research of the US Department of Energy, administered by the Oak Ridge Institute for Science and Education. The authors also acknowledge the support of the US Department of Energy in the form of a grant, DE-FG0592ER61401.

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