Applicazioni biotecnologiche

Transcription

1 Applicazioni biotecnologiche Analisi forense Sintesi di proteine ricombinanti

2 Restriction Fragment Length Polymorphism (RFLP) Polymorphism (more fully genetic polymorphism) refers to the simultaneous occurrence in the population of genomes showing variations at a given position. What is the basis for the polymorphism among the mutant alleles? They possess different mutations that alter the protein function, thus producing changes in phenotype. If we compare the restriction maps or the DNA sequences of these alleles, they too will be polymorphic in the sense that each map or sequence will be different from the others. Although not evident from the phenotype, the wild type may itself be polymorphic. Multiple versions of the wild-type gene may be distinguished by differences in sequence that do not affect their function, and which therefore do not produce phenotypic variants. A population may have extensive polymorphism at the level of genotype. Many different sequence variants may exist for each gene; some of them are evident because they affect the phenotype, but others are hidden because they have no visible effect. A change in a single nucleotide when alleles are compared is called a single nucleotide polymorphism (SNP) occurs every ~1330 bases in the human genome Some polymorphisms in the genome can be detected by comparing the restriction maps of different individuals. The criterion is a change in the pattern of fragments produced by cleavage with a restriction enzyme. When a target site is present in the genome of one individual and absent from another, the extra cleavage in the first genome will generate two fragments corresponding to the single fragment in the second genome. A difference in restriction maps between two individuals is called a restriction fragment length polymorphism (RFLP).

3 Nucleic acids hybridize by base pairing The principle of the hybridization reaction is to expose two single-stranded nucleic acid preparations to each other and then to measure the amount of double-stranded material that forms. A DNA preparation is denatured and the single strands are adsorbed to a filter. A second denatured DNA (or RNA) preparation is added The filter is treated so that the second preparation can adsorb to it only if it is able to base pair with the DNA that was originally adsorbed. Usually the second preparation is radioactively labeled, so that the reaction can be measured as the amount of radioactive label retained by the filter. The extent of hybridization between two single-stranded nucleic acids is determined by their complementarity. Two sequences need not be perfectly complementary to hybridize. If they are closely related but not identical, an imperfect duplex is formed in which base pairing is interrupted at positions where the two single strands do not correspond.

4 DNA fingerprinting DNA fingerprinting analyzes the differences between individuals of the fragments generated by using restriction enzymes to cleave regions that contain short repeated sequences.

5 Restriction Fragment Length Polymorphism (RFLP) Because the restriction map is independent of gene function, a polymorphism at this level can be detected irrespective of whether the sequence change affects the phenotype. Probably very few of the restriction site polymorphisms in a genome actually affect the phenotype. Most involve sequence changes that have no effect on the production of proteins (for example, because they lie between genes). A restriction fragment length polymorphism (basically a SNP that is located in the target site for a restriction enzyme) can be used as a genetic marker in exactly the same way as any other marker. Instead of examining some feature of the phenotype, we directly assess the genotype, as revealed by the restriction map. The existence of RFLPs provides the basis for a technique to establish unequivocal parent-progeny relationships. In cases where parentage is in doubt, a comparison of the RFLP map in a suitable chromosome region between potential parents and child allows absolute assignment of the relationship.

6 DNA cloning A single gene is only a small part of the genome so isolating a DNA fragment containing the gene of interest requires two procedures 1. a DNA library is constructed that contains many thousand of fragments derived from the genome 2. the DNA segment that contains the gene of interest is identified by its sequence A DNA library is a collection of DNA fragments derived from the genome of a particular organism, with each fragment attached to a cloning vector genomic DNA is cleaved in thousand of fragments, and all of them are cloned the total information content of an organism is represented by all fragments in the library Libraries constructed in this way are referred as genomic libraries. Big genome gene under-represented

7 Recovering the information from RNA The central dogma defines the paradigm of molecular biology. Genes are perpetuated as sequences of nucleic acid, but function by being expressed in the form of proteins. Replication is responsible for the inheritance of genetic information. Transcription and translation are responsible for its conversion from one form to another. The perpetuation of nucleic acid may involve either DNA or RNA as the genetic material. Cells use only DNA. Some viruses use RNA, and replication of viral RNA occurs in the infected cell. The expression of cellular genetic information usually is unidirectional. Transcription of DNA generates RNA molecules that can be used further only to generate protein sequences; generally they cannot be retrieved for use as genetic information. Translation of RNA into protein is always irreversible. The restriction to unidirectional transfer from DNA to RNA is not absolute. It is overcome by the retroviruses, whose genomes consist of single-stranded RNA molecules. During the infective cycle, the RNA is converted by the process of reverse transcription into a singlestranded DNA, which in turn is converted into a double-stranded DNA. This duplex DNA becomes part of the genome of the cell, and is inherited like any other gene. So reverse transcription allows a sequence of RNA to be retrieved and used as genetic information.

8 Complementary DNA (cdna) A more specialized and exclusive DNA library can be constructed as to include only those gene that are expressed in certain cells. The mrna from certain cells derived from the organism is extracted and complementary DNAs (cdnas) are produced from the mrna in a multistep reaction catalyzed by reverse transcriptase. The resulting double stranded DNA fragments are then inserted into a suitable vector, creating a population of clones called a cdna library Among the first eukaryotic genes characterized were those encoding globins cloning of those genes was facilitated by making cdna libraries from erythrocytes precursor cells, in which about half of the mrna codes for globins.

9 Nucleic acids hybridize by base pairing The principle of the hybridization reaction is to expose two single-stranded nucleic acid preparations to each other and then to measure the amount of double-stranded material that forms. A DNA preparation is denatured and the single strands are adsorbed to a filter. A second denatured DNA (or RNA) preparation is added The filter is treated so that the second preparation can adsorb to it only if it is able to base pair with the DNA that was originally adsorbed. Usually the second preparation is radioactively labeled, so that the reaction can be measured as the amount of radioactive label retained by the filter. The extent of hybridization between two single-stranded nucleic acids is determined by their complementarity. Two sequences need not be perfectly complementary to hybridize. If they are closely related but not identical, an imperfect duplex is formed in which base pairing is interrupted at positions where the two single strands do not correspond.

10 Library screening After a library has been generated, the challenge is to isolate the gene of interest among the millions of other DNA segments represented in a DNA library. Because each gene has a unique nucleotide sequence, it often can be detected with the aid of a labeled (e.g., radioactive) DNA fragment that is complementary to it, called a probe. Frequently, the limiting step in cloning a gene is finding or generating a complementary strand of nucleic acid to use as probe sometimes, a homologous gene cloned from an other species can be used as probe; alternatively, the protein product of a gene need to be purified.

11 Enhancers contain the same elements A difference between the that are found at promoters enhancer and a typical promoter is presented by the density of regulatory elements. The susceptibility of the SV40 enhancer to damage by mutation shows that a much greater proportion of its sites directly influences its function than is the case with the promoter analyzed in the same way. There is an increase in the density of protein-binding sites. Many of these sites are common elements in promoters; for example, AP1 and the octamer. The specificity of transcription may be controlled by either a promoter or an enhancer. A promoter may be specifically regulated, and a nearby enhancer used to increase the efficiency of initiation; or a promoter may lack specific regulation, but become active only when a nearby enhancer is specifically activated.

12 IL-6 gp130 Protein isolation 3000 mice were injected with IL-6. Liver extracts were prepared. STAT binding sequence was covalently linked to a column. Liver extracts were passed on the column and STAT was affinity purified. Partial sequence was determined.

13 Designing a probe If a protein product of a gene has been purified, probes can be designed and synthesized on the basis of its amino acid sequence and knowledge of the genetic code.

14 Gene isolation by Polymerase Chain Reaction (PCR)

15 DNA sequencing

16 Automated DNA sequencing

17 Recombinant protein synthesis A cloned gene can be used to produce large amounts of his protein product. Understanding the fundamentals of DNA, RNA and protein metabolism in E. coli has made possible to express protein genes to study their protein products. In some cases the protein product is overproduced, representing 10% or more of the cellular proteins such high protein concentration can kill an E. coli cell; gene expression must be limited until a few hours before the planned harvest of the cells. Vectors that have the signals necessary for expression of a cloned gene are often called expression vectors.

18 Recombinant proteins must be purified Uninduced Induced Uninduced Induced Large sc. Pellet Purified Concentr. The number of protein bands decreases as the purification proceeds.