STUDYING GENOMES Studying DNA Enzymes for DNA manipulation

STUDYING GENOMES

Studying DNA

Enzymes for DNA manipulation • Before 1970 s, the only way in which individual genes could be studied was by classical genetics. • Biochemical research provided (in the early 70 s) molecular biologists with enzymes that could be used to manipulate DNA molecules in the test tube. • Molecular biologists adopted these enzymes as tools for manipulating DNA molecules in pre-determined ways, using them to make copies of DNA molecules, to cut DNA molecules into shorter fragments, and to join them together again in combinations that do not exist in nature. • These manipulations form the basis of recombinant DNA technology.

Recombinant DNA technology • The enzymes available to the molecular biologist fall into four broad categories: 1. 2. DNA polymerase – synthesis of new polynucleotides complementary to an existing DNA or RNA template Nucleases – degrade DNA molecules by breaking the phosphodiester bonds • restriction endonucleases (restriction enzyme) – cleave DNA molecules only when specific DNA sequences is encountered 3. 4. Ligases – join DNA molecules together End modification enzymes – make changes to the ends of DNA molecules

source: Brown T. A. , Genomes. 2 nd ed. http: //www. ncbi. nlm. nih. gov/books/NBK 21129/

DNA cloning • DNA cloning (i. e. copying) – logical extension of the ability to manipulate DNA molecules with restriction endonucleases and ligases • vector • DNA sequence that naturally replicates inside bacteria. • It consists of an insert (transgene) and larger sequence serving as the backbone of the vector. • Used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation machinery ribosomal complexes.

Vectors • plasmid • DNA molecule that is separated from, and can replicate independently of, the chromosomal DNA. • Double stranded, usually circular, occurs naturally in bacteria. • Serves as an important tool in genetics and biotechnology labs, where it is commonly used to multiply (clone) or express particular genes. • length of insert: 1 -10 kbp source: wikipedia

Vectors • BAC (bacterial artificial chromosome) • It is a particular plasmid found in E. coli. A typical BAC can carry about 250 kbp (100 -350 kbp). • cosmid • 40 -45 kbp • YAC (yeast artificial chromosome) • 1. 5 -3. 0 Mbp

restriction endonuclease ligase DNA cloning source: Brown T. A. , Genomes. 2 nd ed. http: //www. ncbi. nlm. nih. gov/books/NBK 21129/

PCR – Polymerase chain reaction • DNA cloning results in the purification of a single fragment of DNA from a complex mixture of DNA molecules. • Major disadvantage: it is time-consuming (several days to produce recombinants) and, in parts, difficult procedure. • The next major technical breakthrough (1983) after gene cloning was PCR. • It achieves the amplifying of a short fragment of a DNA molecule in a much shorter time, just a few hours. • PCR is complementary to, not a replacement for, cloning because it has its own limitations: we need to know the sequence of at least part of the fragment.

Mapping genomes

What is it about? • Assigning/locating the specific gene to the particular region at the chromosome and determining the location and relative distances between genes at the chromosome. • There are two types of maps: • genetic linkage map – shows the arrangement of genes (or other markers) along the chromosomes as calculated by the frequency with which they are inherited together • physical map – representation of the chromosomes, providing the physical distance between landmarks on the chromosome, ideally measured in nucleotide bases • The ultimate physical map is the complete sequence itself.

Genetic mapping • use of genetic techniques to construct maps showing the positions of genes • genetic techniques: • cross-breeding experiments • humans: the examination of family histories (pedigrees)

Genetic linkage map • Constructed by observing how frequently two markers (e. g. genes) are inherited together. • Two markers located on the same chromosome can be separated only through the process of recombination. • If they are separated, childs will have just one marker from the pair. • However, the closer the markers are, the more tightly linked they are, and the less likely recombination will separate them. They will tend to be passed together from parent to child. • Recombination frequency provides an estimate of the distance between two markers. Genomes 2, T. A. Brown

• Look at the results of meiosis in a hundred identical cells. • If crossovers never occur then the resulting gametes will have the following genotypes: 200 AB, 200 ab • This is called completed linkage. • But if (as is more likely) crossovers occur between A and B in some of the nuclei, then the allele pairs will not be inherited as single units. Let us say that crossovers occur during 40 of the 100 meioses. The following gametes will result: • 160 AB, 160 ab, 40 a. B • This is called partial linkage. • The recombination frequency depends on the distance between two genes. Genomes 2, T. A. Brown

Genetic markers • A genetic map must show the positions of distinctive features – markers. • Any inherited characteristic that differs among individuals and is easily detectable in the laboratory is a potential genetic marker. • Markers can be • genes • DNA segments that have no known coding function but which inheritance pattern can be followed.

Genetic linkage map • On the genetic maps distances between markers are measured in terms of centimorgans (c. M). • 1 c. M apart – they are separated by recombination 1% of the time • 1 c. M is ROUGHLY equal to physical distance of 1 Mbp in human

Genes as markers • useful but no ideal • For larger genomes (e. g. vertebrates), gene maps are not very detailed (genes are widely spaced out with large gaps between them). • Variations within genes lead to observable changes (e. g. eye color). However, only a fraction of the total number of genes exist in allelic forms that can be distinguished conveniently. • Gene maps are therefore not very comprehensive. We need other types of marker. • Mapped features that are not genes are called DNA markers.

DNA markers • Must be polymorphic, i. e. alternative forms (alleles) must exist among individuals so that they are detectable among different members in family studies. • Most variations occur within introns, have little or no effect on an organism, yet they are detectable at the DNA level and can be used as markers. 1. 2. 3. restriction fragment length polymorphisms (RFLPs) simple sequence length polymorphisms (SSLPs) single nucleotide polymorphisms (SNPs, pronounce “snips”)

RFLPs (restriction fragment length) • Recall that restriction enzymes cut DNA molecules at specific recognition sequences. • This sequence specificity means that treatment of a DNA molecule with a restriction enzyme should always produce the same set of fragments. • This is not always the case with genomic DNA molecules because some restriction sites exist as two alleles, one allele displaying the correct sequence for the restriction site and therefore being cut, and the second allele having a sequence alteration so the restriction site is no longer recognized. source: Brown T. A. , Genomes. 2 nd ed. http: //www. ncbi. nlm. nih. gov/books/NBK 21129/

SSLPs (simple sequence length) • Repeat sequences that display length variations, different alleles contain different numbers of repeat units (i. e. SSLPSs are multiallelic). • variable number of tandem repeat sequences (VNTRs, minisatellites) • repeat unit up to 25 bp in length • simple tandem repeats (STRs, microsatellites) • repeats are shorter, usually di- or tetranucleotide source: Brown T. A. , Genomes. 2 nd ed. http: //www. ncbi. nlm. nih. gov/books/NBK 21129/

SNPs (sigle nucleotide) • Positions in a genome where some individuals have one nucleotide and others have a different nucleotide. • Vast number of SNPs in every genome. • Each SNP could have potentially four alleles, most exist in just two forms. • The value of two-allelic marker (SNP, RFLP) is limited by the high possibility that the marker shows no variability among the members of a family. • The advantages of SNP over RFLP: • they are abundant (human genome: 1. 5 millions of SNPs, 100 000 RFLPs) • easire to type (i. e. easier to detect)

Marker analysis • Value of genetic map – marker analysis • Inherited disease can be located on the map by following the inheritance of a DNA marker present in affected individuals (but absent in unaffected individuals), even though the molecular basis of the disease may not yet be understood nor the responsible gene identified. • This represent a cornerstone of testing for genetic diseases.

Physical maps • A map generated by genetic techniques is rarely sufficient for directing the sequencing phase of a genome project. 1. The resolution of a genetic map depends on the number of crossovers that have been scored. 2. Genetic maps have limited accuracy. • Before large-scale sequencing begins (see next lecture), a genetic map must be checked and supplemented by alternative mapping procedure.

Physical mapping • A plethora of physical mapping techniques has been developed to address this problem, the most important being 1. 2. 3. Restriction mapping Fluorescent in situ hybridization (FISH) Sequence tagged site (STS) mapping see more at Genomes 2, Brown, http: //www. ncbi. nlm. nih. gov/books/NBK 21116/

Genetic and physical map - compared • Saccharomyces cerevisiae chromosome III • physical map obtained by DNA sequencing • the order of the upper two markers (glk 1 and cha 1) is incorrect on the genetic map • there also differences in the relative positioning of other pairs of markers Oliver SG et al. , Nature, 357, 38– 46

more at http: //www. informatics. jax. org/silver/chapters/7 -1. shtml Genome maps relative locations of markers are established by following inheritance patterns visual appearance of a chromosome when stained and examined under a microscope the order and spacing of the markers, measured in base pairs sequence map source: Talking glossary of genetic terms, http: //www. genome. gov/glossary/

NCBI human Genome Resources • http: //www. ncbi. nlm. nih. gov/genome/guide/human/