Genomes & Biotechnology

1. Genomes & Biotechnology

2. Key Concepts • There Are Powerful Methods for Sequencing Genomes and Analyzing Gene Products • Prokaryotic & Eukaryotic Genomes have several things in common, but some key differences • The Human Genome Sequence Has Many Applications

3. Key Concepts • Recombinant DNA Can Be Made in the Laboratory • DNA Can Genetically Transform Cells and Organisms • Genes and Gene Expression Can Be Manipulated • Biotechnology Has Wide Applications

4. There Are Powerful Methods for Sequencing Genomes and Analyzing Gene Products • The Human Genome Project was proposed in 1986 to determine the normal sequence of all human DNA. • The publicly funded effort was aided and complemented by privately funded groups. • Methods used were first developed to sequence prokaryotes and simple eukaryotes. (Sanger Method) • The fragment sequences are put together using larger, overlapping fragments. Strand to be sequenced Each flask has a different terminating nucleotide Fragment lengths determined by gel electrophoresis Replication products

5. There Are Powerful Methods for Sequencing Genomes and Analyzing Gene Products • Next-generation DNA sequencing methods (Next gen) developed in the 1990’s use DNA replication and the polymerase chain reaction (PCR). These enabled the project to be finished much quicker than originally anticipated, 2003.

6. Figure 12.1 DNA Sequencing (Part 1) • One approach to next-generation DNA sequencing: • DNA is cut into 100 bp fragments. • DNA is denatured by heat, and each single strand then acts a template for synthesis. • Each fragment is attached to adapter sequences and then to supports. • Fragments are then amplified by PCR. • Amplified DNA attached to a solid substrate is ready for sequencing: • Fragments are denatured and primers, DNA polymerase, and fluorescently labeled nucleotides are added. • DNA is replicated by adding one nucleotide at a time. • Fluorescent color of the particular nucleotide is detected as it is added, indicating the sequence of the DNA.

7. There Are Powerful Methods for Sequencing Genomes and Analyzing Gene Products • Determining sequences is possible because original DNA fragments are overlapping. • Example: A 10 bp fragment cut three different ways yields • TG, ATG, and CCTAC • AT, GCC, and TACTG • CTG, CTA, and ATGC • The correct sequence is ATGCCTACTG.

8. Figure 12.2 Arranging DNA Sequences For genome sequencing the fragments are called “reads.”Using complex mathematics and computer programs further increases the speed at which “reads” are processed. A field known as bioinformatics.

9. There Are Powerful Methods for Sequencing Genomes and Analyzing Gene Products • The power of this method derives from the fact that: • • It is fully automated and miniaturized. • • Millions of different fragments are sequenced at the same time. This is called massively parallel sequencing. • • It is an inexpensive way to sequence large genomes.

10. There Are Powerful Methods for Sequencing Genomes and Analyzing Gene Products • It is one thing to know the order of the nucleotide bases, it is another to know what it all means…. • In functional genomics, sequences identify the functions of various parts: • Open reading frames—the coding regions of the genes, recognized by start and stop codons for translation, and sequences indicating location of introns • Amino acid sequences of proteins • Regulatory sequences—promoters and terminators for transcription • RNA genes, including rRNA, tRNA, small nuclear RNA, and microRNA genes • Other noncoding sequences in various categories • Comparative genomics compares a newly sequenced genome with sequences from other organisms. • It provides information about function of sequences and can trace evolutionary relationships.

11. There Are Powerful Methods for Sequencing Genomes and Analyzing Gene Products • The proteome is the total of the proteins produced by an organism—more complex than its genome. • Many genes encode for more than one protein, through alternative splicing and posttranslational modifications. • Proteomics seeks to identify and characterize all of the expressed proteins.

12. There Are Powerful Methods for Sequencing Genomes and Analyzing Gene Products • The metabolome is the description of all of the metabolites of a cell or organism: • Primary metabolites are involved in normal processes, such as in pathways like glycolysis. Also includes hormones and other signaling molecules. • Secondary metabolites are often unique to particular organisms or groups. • Examples: Antibiotics made by microbes, and chemicals made by plants for defense. • Metabolomics aims to describe the metabolome of a tissue or organism under particular environmental conditions. • Analytical instruments can separate molecules with different chemical properties, and other techniques can identify them. • Measurements can be related to physiological states.

13. Figure 12.5 Genomics, Proteomics, and Metabolomics

14. Prokaryotic & Eukaryotic Genomes have several things in common, but some key differences • Comparing genomes of prokaryotes and eukaryotes: • Certain genes are present in all organisms (universal genes); and some universal gene segments are present in many organisms. • This suggests that a minimal set of DNA sequences is common to all cells.

15. Prokaryotic & Eukaryotic Genomes have several things in common, but some key differences • Efforts to define a minimal genome of life involve computer analysis of genomes, the study of the smallest known genome (M. genitalium), and using transposons as mutagens. • Transposons can insert into genes at random; the mutated bacteria are tested for growth and survival, and DNA is sequenced.

16. Prokaryotic & Eukaryotic Genomes have several things in common, but some key differences • Transposons are of two main types in eukaryotes: • Retrotransposons(Class I) make RNA copies of themselves, which are copied into DNA and inserted in the genome. • LTR retrotransposons have long terminal repeats of DNA sequences • Non-LTR retrotransposons do not have LTR sequences DNA transposons (Class II) do not use RNA intermediates. • They are excised from the original location and inserted at a new location without being replicated.

17. Prokaryotic & Eukaryotic Genomes have several things in common, but some key differences • Transposons (or transposable elements) are DNA segments that can move from place to place in the genome. • They can move from one piece of DNA (such as a chromosome), to another (such as a plasmid). • If a transposon is inserted into the middle of a gene, it will be transcribed and result in abnormal proteins. • If a small transposon is duplicated and the 2 copies are then separated by host genes, the whole complex can be carried to other locations within the genome. This can result in multiple copies of a gene.

18. Prokaryotic & Eukaryotic Genomes have several things in common, but some key differences • A group of closely related genes are called gene families . • These arose over evolutionary time when different copies of genes underwent separate mutations. • For example: Genes encoding the globin proteins in hemoglobin and myoglobin all arose from a single common ancestral gene. • Many gene families include nonfunctional pseudogenes (Ψ), resulting from mutations that cause a loss of function, rather a new one. • A pseudogene may simply lack a promoter, and thus fail to be transcribed, or a recognition site, needed for the removal of an intron.

19. Prokaryotic & Eukaryotic Genomes have several things in common, but some key differences • Eukaryotic genomes have repetitive DNA sequences: • Highly repetitive sequences—short sequences (< 100 bp) repeated thousands of times in tandem; not transcribed • Short tandem repeats (STRs) of 1–5 bp are scattered around the genome and can be used in DNA fingerprinting. • Moderately repetitive sequences are repeated 10–1,000 times. • Includes the genes for tRNAs and rRNAs • Single copies of the tRNA and rRNA genes are inadequate to supply large amounts of these molecules needed by cells, so genome has multiple copies in clusters • Most moderately repeated sequences are transposons.

20. Table 12.3 Types of Sequences in Eukaryotic Genomes

21. Prokaryotic & Eukaryotic Genomes have several things in common, but some key differences

22. Prokaryotic & Eukaryotic Genomes have several things in common, but some key differences • Several model organisms have been studied and used extensively. • Model organisms are easy to grow and study in a laboratory, their genetics are well studied, and their characteristics represent a larger group of organisms.

23. Prokaryotic & Eukaryotic Genomes have several things in common, but some key differences • Prokaryotes can be identified by their growth in culture, but DNA can also be isolated directly from environmental samples. • DNA can then be cloned for “libraries” or amplified and sequenced to detect known and unknown organisms. • E. coli is often used to “store” the library of genes.

24. The Human Genome Sequence Has Many Applications • By 2010 the complete haploid genome sequence was completed for more than ten individuals. • Now scientists are working on the 1000 project: • The 1000 Genomes Project is an international collaboration to produce an extensive public catalog of human genetic variation, including SNPs and structural variants, and their haplotype contexts. This resource will support genome-wide association studies and other medical research studies.The genomes of about 2500 unidentified people from about 25 populations around the world will be sequenced using next-generation sequencing technologies. The results of the study will be freely and publicly accessible to researchers worldwide. • http://www.1000genomes.org/ • The average person can also explore facets of their own DNA for as little as a $100.

25. The Human Genome Sequence Has Many Applications • Some interesting facts about the human genome: • Protein-coding genes make up about 24,000 genes, less than 2 percent of the 3.2 billion base pair human genome. • Each gene must code for several proteins, and posttranscriptional mechanisms (e.g., alternative splicing) must account for the observed number of proteins in humans. • An average gene has 27,000 base pairs, but size varies greatly as does the size of the proteins. • All human genes have many introns. • 3.5 percent of the genome is functional but noncoding—have roles in gene regulation (microRNAs) or chromosome structure. • Over 50 percent of the genome is transposons and other repetitive sequences. • Most of the genome (97 percent) is the same in all people. • Chimpanzees share 95 percent of the human genome.

26. Figure 12.9 Functions of the Eukaryotic Genome

27. Figure 12.12 Evolution of the Genome

28. The Human Genome Sequence Has Many Applications • Rapid genotyping technologies are being used to understand the complex genetic basis of diseases such as diabetes, heart disease, and Alzheimer’s disease. • “Haplotype maps” are based on single nucleotide polymorphisms (SNPs)—DNA sequence variations that involve single nucleotides. • SNPs are point mutations in a DNA sequence.

29. The Human Genome Sequence Has Many Applications • SNPs that differ are not all inherited as independent alleles. • A set of SNPs that are close together on a chromosome are inherited as a linked unit. • A piece of chromosome with a set of linked SNPs is called a haplotype. • Analyses of human haplotypes have shown that there are, at most, 500,000 common variations. • .

30. The Human Genome Sequence Has Many Applications • Technologies to analyze SNPs in an individual genome include next-generation sequencing methods and DNA microarrays. • A DNA microarray detects DNA or RNA sequences that are complementary to and hybridize with an oligonucleotide probe. • The aim is to find out which SNPs are associated with specific diseases and identify alleles that contribute to disease.

31. Figure 12.13 SNP Genotyping and Disease

32. The Human Genome Sequence Has Many Applications • Genetic variation can affect an individual’s response to a particular drug. • A variation could make an drug more or less active in an individual. • Pharmacogenomics studies how the genome affects the response to drugs. • This makes it possible to predict whether a drug will be effective, with the objective of personalizing drug treatments.

33. The Human Genome Sequence Has Many Applications • DNA fingerprinting refers to a group of techniques used to identify individuals by their DNA. • Short tandem repeat (STR) analysis is most common. • When several different STR loci are analyzed, a unique pattern becomes apparent. • Can be used for questions of paternity and in crime investigation

34. Recombinant DNA Can Be Made in the Laboratory • It is possible to modify organisms with genes from other, distantly related organisms. • Recombinant DNA is a DNA molecule made in the laboratory that is derived from at least two genetic sources. • Three key tools: • Restriction enzymes for cutting DNA into fragments • Gel electrophoresis for analysis and purification of DNA fragments • DNA ligase for joining DNA fragments together in new combinations

35. Recombinant DNA Can Be Made in the Laboratory • Restriction enzymes recognize a specific DNA sequence called a recognition sequence or restriction site. • 5′…….GAATTC……3′ • 3′…….CTTAAG……5′ • Each sequence forms a palindrome: the opposite strands have the same sequence when read from the 5′ end. • Some restriction enzymes create a blunt cut in DNA leaving a short sequence of single-stranded DNA at each end. • Staggered cuts result in overhangs, or “sticky ends;” straight cuts result in “blunt ends.” • Sticky ends can bind complementary sequences on other DNA molecules.

36. Recombinant DNA Can Be Made in the Laboratory • DNA fragments cut by enzymes can be separated by gel electrophoresis. • Negatively charged DNA fragments move towards the positive end. • Smaller fragments move faster than larger ones. • DNA fragments separate and give three types of information: • The number of fragments • The sizes of the fragments • The relative abundance of the fragments, indicated by the intensity of the band

37. Recombinant DNA Can Be Made in the Laboratory • After separation on a gel, a specific DNA sequence can be found with a complementary single-stranded probe. • The gel region can be cut out and the DNA fragment removed. • The purified DNA can be analyzed by sequence or used to make recombinant DNA. • With restriction enzymes to cut fragments and DNA ligase to combine them, new recombinant DNA can be made.

38. Concept 13.2 DNA Can Genetically Transform Cells and Organisms • Recombinant DNA technology can be used to clone (make identical copies) genes. • Transformation: Recombinant DNA is cloned by inserting it into host cells (transfection if host cells are from an animal). • The altered host cell is called transgenic. • Usually only a few cells exposed to recombinant DNA are actually transformed. • To determine which of the host cells are transgenic, the recombinant DNA includes selectable marker genes, such as genes that confer resistance to antibiotics. (Refer to bacterial transformation lab)

39. Concept 13.2 DNA Can Genetically Transform Cells and Organisms • Selectable markers are a type of reporter gene—a gene whose expression is easily observed. • Green fluorescent protein, which normally occurs in a jellyfish, emits visible light when exposed to UV light. • The gene for this protein has been isolated and incorporated into vectors as a reporter gene.

40. DNA Can Genetically Transform Cells and Organisms • Methods for inserting the recombinant DNA into a cell: • Cells may be treated with chemicals to make plasma membranes more permeable—DNA diffuses in. (CaCl2) • Electroporation—a short electric shock creates temporary pores in membranes, and DNA can enter. • Biological Vector - Viruses and bacteria can be altered to carry recombinant DNA into cells. • Mechanical Vector - “Gene guns” can “shoot” the host cells with particles of DNA. • Transgenic animals can be produced by injecting recombinant DNA into the nuclei of fertilized eggs.