Fundamentals of Bioinformatics - Ogurtsov A.N. 2013

Basics of Bioinformatics
Genomes and Proteomes
Genome Mapping

Before the advent of Genome Analysis technologies, the genetic knowledge base of an Organism typically consisted of relatively low-resolution chromosome maps and physical maps of genes producing known mutant phenotypes. Starting from genetic linkage maps, the construction of whole-genome molecular maps generally proceeds through several stages of successively increasing resolution (Figure 40).

A genetic map is a representation of the relative distances between genes, estimated on The basis of measured recombination frequencies between those genes.

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Figure 40 - Hierarchy of whole-genome mapping: a - cytogenetic map; b - high-resolution genetic map; c - physical Gene map; d - nucleotide DNA sequence

Genetic mapping is The process of determining the chromosomal assignment of genes and assigning them genetic distances relative to other (already known) genes. Genome genetic maps are constructed using data from genetic crosses or, in the case of humans, pedigree analysis. Genetic crosses make it possible to establish the locations of genetic markers on Chromosomes and determine the genetic distance between them. A genetic marker is a DNA segment with a known Location. It can be an allele with a known location that determines a specific trait; a distinct morphological feature of a chromosome, such as a constriction (morphological marker); or polymorphic DNA fragments (molecular markers). Genetic markers serve as reference points for gene mapping. Previously, genes themselves were used as experimental markers in constructing genetic maps.

Today, genetic maps are constructed using another type of genetic marker—DNA markers. The latter are genetic markers detected using molecular tools that operate directly on the DNA itself, rather than on the gene product or derived phenotype. Four MAIN TYPES OF DNA markers are used in constructing The Human Genome map:

1) RFLP - Restriction Fragment Length Polymorphism;

2) VNTR - Variable Number of Tandem Repeat, also referred to as a minisatellite;

3) STR - Short Tandem Repeat, also referred to as a microsatellite;

4) SNP - Single Nucleotide Polymorphism. Using DNA Microarrays (see section 11.4), hundreds of SNPs can be printed simultaneously.

Variable Number of Tandem Repeat loci (VNTRs) (minisatellites). VNTRs contain segments 10-100 bp in length, repeated a variable number of times. In different individuals, VNTRs built on the same motif may contain different numbers of repeats. Differences in the lengths of these fragments can be used as genetic markers. The inheritance of VNTRs can be tracked and linked to a specific phenotype. VNTRs were the first genetic data used for personal identification in forensics, as well as in paternity disputes. This method was termed DNA fingerprinting. Previously, VNTRs were studied based on Restriction Fragment Length Polymorphisms (RFLPs).

VNTRs almost always contain multiple restriction sites for the same restriction enzyme, allowing them to be precisely cleaved. The results can be separated on a gel and subjected to Southern blotting (see [7], section 11.4).

It should be borne in mind that VNTRs (Variable Number of Tandem Repeat) are genomic features, whereas RFLPs (Restriction Fragment Length Polymorphism) represent an artificial mixture of restriction fragments generated in the laboratory to identify VNTRs.

Currently, PCR (Polymerase Chain Reaction) is increasingly used to measure VNTR length, a method that has almost entirely replaced The Use of Restriction Enzymes in this field.

Short Tandem Repeat (STR) polymorphisms (microsatellites) are segments of 2-5 bp repeated A large number of times (10-30). There are many advantages to using STRs, one of which is their fairly uniform distribution across The Genome.

Microsatellites typically lie outside of expressed genes (with exceptions such as trinucleotide repeats in the genes for Huntington's disease (see section 6.6) and certain other disorders).

A panel of microsatellite markers significantly simplifies gene identification.

Various complementary mapping techniques work more precisely with DNA, thereby shortening the gene identification process.

A contig, or contiguous clone map, is a series of overlapping chromosomal DNA clones in a known order (see section 2.3) maintained in Cells as YACs (Yeast Artificial Chromosomes) or BACs (Bacterial Artificial Chromosomes). Such a map provides an excellent representation of the genome.

In YACs, human DNA is integrated into small additional yeast Cell chromosomes. Such a chromosome can contain up to 10⁶ bp, and the entire human genome can be accommodated in 10,000 YACs.

In bacterial artificial chromosomes (BACs), DNA is inserted into an E. coli plasmid. A plasmid is a separate, small, circular, double-stranded DNA molecule that acts as an adjunct to the main genome. A bacterial artificial chromosome can carry up to 250,000 bp.

Although this capacity is smaller than that of YACs, BACs are preferred because they are more stable and easier to handle.

A sequence tagged site (STS) is a short, sequenced DNA segment, typically 200-600 bp in length, located in a strictly defined region of the genome. It does not need to be polymorphic. An STS can be mapped onto the genome using PCR and cells containing a contiguous clone map.

One type of STS originates from Expressed Sequence Tags (ESTs), which are short cDNA fragments (see sections 1.1 and 11.3).

cDNA—complementary DNA—is a sequence derived from the mRNA of an expressed gene using the enzyme Reverse Transcriptase (see [7], section 9.4).

EST sequences contain only exons spliced together into a protein-coding sequence. cDNA can be mapped to a chromosome using the FISH (Fluorescence In Situ Hybridization) method or localized within a contig map.

If an organism is being studied for which the complete genome sequence is not known, but for which a complete contig map for all chromosomes is available, STS markers tightly linked to the gene of interest can be identified and subsequently localized on the contig map.