A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.

AFLP: a new technique for DNA fingerprinting.

Simple and reproducible fingerprints of complex genomes can be generated using single arbitrarily chosen primers and the polymerase chain reaction (PCR). No prior sequence information is required. The method, arbitrarily primed PCR (AP-PCR), involves two cycles of low stringency amplification followed by PCR at higher stringency. We show that strains can be distinguished by comparing polymorphisms in genomic fingerprints. The generality of the method is demonstrated by application to twenty four strains from five species of Staphylococcus, eleven strains of Streptococcus pyogenes and three varieties of Oryza sativa (rice).

Fingerprinting genomes using PCR with arbitrary primers

Effective methods are needed to identify and isolate those genes that are differentially expressed in various cells or under altered conditions. This report describes a method to separate and clone individual messenger RNAs (mRNAs) by means of the polymerase chain reaction. The key element is to use a set of oligonucleotide primers, one being anchored to the polyadenylate tail of a subset of mRNAs, the other being short and arbitrary in sequence so that it anneals at different positions relative to the first primer. The mRNA subpopulations defined by these primer pairs were amplified after reverse transcription and resolved on a DNA sequencing gel. When multiple primer sets were used, reproducible patterns of amplified complementary DNA fragments were obtained that showed strong dependence on sequence specificity of either primer.

https://science.sciencemag.org/content/sci/257/5072/967.full.pdf

Differential Display of Eukaryotic Messenger RNA by Means of the Polymerase Chain Reaction

A retroviral vector system based on the human immunodeficiency virus (HIV) was developed that, in contrast to a murine leukemia virus-based counterpart, transduced heterologous sequences into HeLa cells and rat fibroblasts blocked in the cell cycle, as well as into human primary macrophages. Additionally, the HIV vector could mediate stable in vivo gene transfer into terminally differentiated neurons. The ability of HIV-based viral vectors to deliver genes in vivo into nondividing cells could increase the applicability of retroviral vectors in human gene therapy.

http://science.sciencemag.org/content/sci/272/5259/263.full.pdf

In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector

We developed bulked segregant analysis as a method for rapidly identifying markers linked to any specific gene or genomic region. Two bulked DNA samples are generated from a segregating population from a single cross. Each pool, or bulk, contains individuals that are identical for a particular trait or genomic region but arbitrary at all unlinked regions. The two bulks are therefore genetically dissimilar in the selected region but seemingly heterozygous at all other regions. The two bulks can be made for any genomic region and from any segregating population. The bulks are screened for differences using restriction fragment length polymorphism probes or random amplified polymorphic DNA primers. We have used bulked segregant analysis to identify three random amplified polymorphic DNA markers in lettuce linked to a gene for resistance to downy mildew. We showed that markers can be reliably identified in a 25-centimorgan window on either side of the targeted locus. Bulked segregant analysis has several advantages over the use of near-isogenic lines to identify markers in specific regions of the genome. Genetic walking will be possible by multiple rounds of bulked segregation analysis; each new pair of bulks will differ at a locus identified in the previous round of analysis. This approach will have widespread application both in those species where selfing is possible and in those that are obligatorily outbreeding.

https://www.pnas.org/content/pnas/88/21/9828.full.pdf

Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations.

Molecular genetic maps are commonly constructed by analyzing the segregation of restriction fragment length polymorphisms (RFLPs) among the progeny of a sexual cross. Here we describe a new DNA polymorphism assay based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence. These polymorphisms, simply detected as DNA segments which amplify from one parent but not the other, are inherited in a Mendelian fashion and can be used to construct genetic maps in a variety of species. We suggest that these polymorphisms be called RAPD markers, after Random Amplified Polymorphic DNA.

/pdf/dna-polymorphisms-amplified-by-arbitrary-primers-are-useful-3dtvzw1rfi.pdf

DNA polymorphisms amplified by arbitrary primers are useful as genetic markers

The complete nucleotide sequence of two human T-cell leukaemia type III (HTLV-III) proviral DNAs each have four long open reading frames, the first two corresponding to the gag and pol genes. The fourth open reading frame encodes two functional polypeptides, a large precursor of the major envelope glycoprotein and a smaller protein derived from the 3′-terminus long open reading frame analogous to the long open reading frame (lor) product of HTLV-I and -II.

Complete nucleotide sequence of the AIDS virus, HTLV-III

The human dopamine D4 receptor contains a novel polymorphism within the putative third cytoplasmic loop of the protein. The polymorphism is characterized by a varying number of direct imperfect 48-bp repeats in the gene. Pharmacological characterization has suggested that this receptor is the site through which the atypical neuroleptic clozapine exerts its antipsychotic action and that some polymorphic variants display different pharmacological properties. Further analysis of the repeat region using innovative technologies indicates that the alleles vary not only in the number of repeats (2-8 or 10 repeat units) but also in the sequence of the repeats and the order in which they appear. In 178 unrelated chromosomes we have identified 19 different repeats in 25 different haplotypes coding for 18 different predicted amino acid sequences, making this one of the most variable functional proteins currently described.

A hypervariable segment in the human dopamine receptor D4 (DRD4) gene

The dopamine D4 receptor gene (DRD4) has an expressed polymorphism in the third exon that may have functional relevance. The polymorphism exists at two levels. At the higher level there is an imperfect tandem repeat of 48 base pairs (bp) coding for 16 amino acids; alleles have been identified with 2 (32 amino acids) to 10 (160 amino acids) repeats. The imperfect nature of the repeats is responsible for a more subtle level of variation since alleles with the same number of repeats can differ in the exact sequences or in the order of the variants of the 48-bp unit. We have undertaken a global survey of this expressed polymorphism as one approach to understanding the evolutionary significance and origins of the polymorphism as well as understanding what selective forces, if any, may be operating at this locus. As the first step, we have determined the repeat number genotype of the DRD4 repeat polymorphism in 1,327 individuals from 36 different populations. The allele frequencies differ considerably among the different populations. The 4-repeat allele was the most prevalent (global mean allele frequency = 64.3%) and appeared in every population with a frequency ranging from 0.16 to 0.96. The 7-repeat allele was the second most common (global mean = 20.6%), appearing quite frequently in the Americas (mean frequency = 48.3%) but only occasionally in East and South Asia (mean frequency = 1.9%). The 2-repeat allele was the third most common (global mean frequency = 8.2%) and was quite frequent in East and South Asia (mean frequency = 18.1%) while uncommon in the Americas (mean frequency = 2.9%) and Africa (mean frequency = 1.7%). The universality of the polymorphism with only three common repeat-number alleles (4, 7, and 2) indicates that the polymorphism is ancient and arose before the global dispersion of modern humans. The diversity of actual allele frequencies for this expressed polymorphism among different populations emphasizes the importance of population considerations in the design and interpretation of any association studies carried out with this polymorphism.

The world-wide distribution of allele frequencies at the human dopamine D4 receptor locus.

We have determined the nucleotide sequence of the yeast ILV2 gene which codes for the amino acid biosynthetic enzyme acetolactate synthase (ALS). ALS has recently been shown to be the target in bacteria, yeast and plants, of the potent new herbicide sulfometuron methyl. The coding sequence for the ILV2 polypeptide contains 2061 base pairs. Comparison of deduced amino acid sequences indicates considerable conservation between the yeast protein and the large subunits of the E. coli ALS II and ALS III isozymes. A major distinction between the three proteins is the presence of an additional 90 amino acids at the amino terminal of the yeast protein. The amino acid sequence in this region shows similarities to yeast mitochondrial transit sequences and may function as such, since yeast ALS is localized in the mitochondria. Consensus sequences for initiation and termination of transcription that are consistent with the ends of the ILV2 mRNA, as well as general amino acid control regulatory sequences have been identified.

Kenneth J. Livak

Papers

DNA polymorphisms amplified by arbitrary primers are useful as genetic markers

Complete nucleotide sequence of the AIDS virus, HTLV-III

A hypervariable segment in the human dopamine receptor D4 (DRD4) gene

The world-wide distribution of allele frequencies at the human dopamine D4 receptor locus.

Nucleotide sequence of the yeast ILV2 gene which encodes acetolactate synthase