Biotechnology and its fundamental final (1).ppt
- 2. DNA • DNA (or deoxyribonucleic acid) is the molecule that carries the genetic information in all cellular forms of life and some viruses. • It belongs to a class of molecules called the nucleic acids, which are polynucleotides - that is, long chains of nucleotides.
- 3. Each nucleotide consists of three components: • a nitrogenous base: • cytosine (C), • guanine (G), • adenine (A) or • thymine (T) • a five-carbon sugar molecule (deoxyribose in the case of DNA) a phosphate molecule
- 4. • The backbone of the polynucleotide is a chain of sugar and phosphate molecules. • Each of the sugar groups in this sugar-phosphate backbone is linked to one of the four nitrogenous bases.
- 5. • DNA's ability to store - and transmit - information lies in the fact that it consists of two polynucleotide strands that twist around each other to form a double-stranded helix. • The bases link across the two strands in a specific manner using hydrogen bonds: cytosine (C) pairs with guanine (G), and adenine (A) pairs with thymine (T).
- 7. • The double helix of the complete DNA molecule resembles a spiral staircase, with two sugar phosphate backbones and the paired bases in the centre of the helix. • This structure explains two of the most important properties of the molecule. • First, it can be copied or 'replicated', as each strand can act as a template for the generation of the complementary strand. • Second, it can store information in the linear sequence of the nucleotides along each strand.
- 9. • It is the order of the bases along a single strand that constitutes the genetic code. • The fourletter 'alphabet' of A, T, G and C forms 'words' of three letters called codons. Individual codons code for specific amino acids. • A gene is a sequence of nucleotides along a DNA strand - with 'start' and 'stop' codons and other regulatory elements - that specifies a sequence of amino acids that are linked together to form a protein.
- 10. • So, for example, the codon AGC codes for the amino acid serine, and the codon ACC codes for the amino acid threonine.
- 11. There are a two points to note about the genetic code: • It is universal. All life on Earth uses the same code (with a few minor exceptions). • It is degenerate. Each amino acid can be coded for by more than one codon. • For example, AGC and ACC both code for the amino acid serine.
- 12. A codon table sets out how the triplet codons code for specific amino acids.
- 13. DNA replication • The enzyme helicase breaks the hydrogen bonds holding the two strands together, and both strands can then act as templates for the production of the opposite strand. • The process is catalysed by the enzyme DNA polymerase, and includes a proofreading mechanism.
- 15. Genes • The gene is the basic physical and functional unit of heredity. • It consists of a specific sequence of nucleotides at a given position on a given chromosome that codes for a specific protein (or, in some cases, an RNA molecule).
- 16. Genes consist of three types of nucleotide sequence • coding regions, called exons, which specify a sequence of amino acids • non-coding regions, called introns, which do not specify amino acids regulatory sequences, which play a role in determining when and where the protein is made (and how much is made) • A human being has 20,000 to 25,000 genes located on 46 chromosomes (23 pairs). These genes are known, collectively, as the human genome.
- 17. Chromosomes • Eukaryotic chromosomes • The label eukaryote is taken from the Greek for 'true nucleus', and eukaryotes (all organisms except viruses, Eubacteria and Archaea) are defined by the possession of a nucleus and other membrane-bound cell organelles.
- 18. • The nucleus of each cell in our bodies contains approximately 1.8 metres of DNA in total, although each strand is less than one millionth of a centimetre thick. • This DNA is tightly packed into structures called chromosomes, which consist of long chains of DNA and associated proteins.
- 19. • In eukaryotes, DNA molecules are tightly wound around proteins - called histone proteins - which provide structural support and play a role in controlling the activities of the genes. • A strand 150 to 200 nucleotides long is wrapped twice around a core of eight histone proteins to form a structure called a nucleosome.
- 20. • The histone octamer at the centre of the nucleosome is formed from two units each of histones H2A, H2B, H3, and H4. • The chains of histones are coiled in turn to form a solenoid, which is stabilised by the histone H1. • Further coiling of the solenoids forms the structure of the chromosome proper.
- 21. • Each chromosome has a p arm and a q arm. • The p arm (from the French word 'petit', meaning small) is the short arm, and the q arm (the next letter in the alphabet) is the long arm. • In their replicated form, each chromosome consists of two chromatids.
- 23. • The chromosomes - and the DNA they contain - are copied as part of the cell cycle, and passed to daughter cells through the processes of mitosis and meiosis. • Human beings have 46 chromosomes, consisting of 22 pairs of autosomes and a pair of sex chromosomes: two X sex chromosomes for females (XX) and an X and Y sex chromosome for males (XY). • One member of each pair of chromosomes comes from the mother (through the egg cell); one member of each pair comes from the father (through the sperm cell).
- 24. A photograph of the chromosomes in a cell is known as a karyotype. The autosomes are numbered 1-22 in decreasing size order.
- 25. Prokaryotic chromosomes • The prokaryotes (Greek for 'before nucleus' - including Eubacteria and Archaea) lack a discrete nucleus, and the chromosomes of prokaryotic cells are not enclosed by a separate membrane.
- 26. • Most bacteria contain a single, circular chromosome. (There are exceptions: some bacteria - for example, the genus Streptomyces - possess linear chromosomes, and Vibrio cholerae, the causative agent of cholera, has two circular chromosomes.) • The chromosome - together with ribosomes and proteins associated with gene expression - is located in a region of the cell cytoplasm known as the nucleoid.
- 27. • The genomes of prokaryotes are compact compared with those of eukaryotes, as they lack introns, and the genes tend to be expressed in groups known as operons. • The circular chromosome of the bacterium Escherichia coli consists of a DNA molecule approximately 4.6 million nucleotides long. • In addition to the main chromosome, bacteria are also characterised by the presence of extrachromosomal genetic elements called plasmids. • These relatively small circular DNA molecules usually contain genes that are not essential to growth or reproduction.
- 28. Overview: The DNA Toolbox • Sequencing of the genomes of more than 7,000 species was under way in 2010 • DNA sequencing has depended on advances in technology, starting with making recombinant DNA • In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule
- 29. • Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes • DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products • An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes © 2011 Pearson Education, Inc.
- 30. DNA cloning yields multiple copies of a gene or other DNA segment • To work directly with specific genes, scientists prepare well-defined segments of DNA in identical copies, a process called DNA cloning © 2011 Pearson Education, Inc.
- 31. DNA Cloning and Its Applications: A Preview • Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids • Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome • Cloned genes are useful for making copies of a particular gene and producing a protein product © 2011 Pearson Education, Inc.
- 32. • Gene cloning involves using bacteria to make multiple copies of a gene • Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell • Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA • This results in the production of multiple copies of a single gene © 2011 Pearson Education, Inc.
- 33. Figure 20.2 Bacterium Bacterial chromosome Plasmid 2 1 3 4 Gene inserted into plasmid Cell containing gene of interest Recombinant DNA (plasmid) Gene of interest Plasmid put into bacterial cell DNA of chromosome (“foreign” DNA) Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of interest Protein expressed from gene of interest Protein harvested Copies of gene Basic research and various applications Basic research on protein Basic research on gene Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hormone treats stunted growth
- 34. Figure 20.2a Bacterium Bacterial chromosome Plasmid 2 1 Gene inserted into plasmid Cell containing gene of interest Recombinant DNA (plasmid) Gene of interest Plasmid put into bacterial cell DNA of chromosome (“foreign” DNA) Recombinant bacterium
- 35. Figure 20.2b Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of interest Protein expressed from gene of interest Protein harvested Copies of gene Basic research and various applications 3 4 Basic research on protein Basic research on gene Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hormone treats stunted growth
- 36. Using Restriction Enzymes to Make Recombinant DNA • Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites • A restriction enzyme usually makes many cuts, yielding restriction fragments • The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends.” © 2011 Pearson Education, Inc.
- 37. © 2011 Pearson Education, Inc. Animation: Restriction Enzymes Right-click slide / select “Play”
- 38. • Sticky ends can bond with complementary sticky ends of other fragments • DNA ligase is an enzyme that seals the bonds between restriction fragments © 2011 Pearson Education, Inc.
- 39. Figure 20.3-1 Restriction enzyme cuts sugar-phosphate backbones. Restriction site DNA 5 5 5 5 5 5 3 3 3 3 3 3 1 Sticky end GAATTC CTTAAG
- 40. Figure 20.3-2 One possible combination DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. Restriction enzyme cuts sugar-phosphate backbones. Restriction site DNA 5 5 5 5 5 5 5 5 5 5 5 5 5 5 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 1 Sticky end GAATTC CTTAAG G G G G AATT C AATT C C TTAA C TTAA
- 41. Figure 20.3-3 Recombinant DNA molecule One possible combination DNA ligase seals strands DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. Restriction enzyme cuts sugar-phosphate backbones. Restriction site DNA 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 3 1 Sticky end GAATTC CTTAAG G G G G AATT C AATT C C TTAA C TTAA
- 42. Cloning a Eukaryotic Gene in a Bacterial Plasmid • In gene cloning, the original plasmid is called a cloning vector • A cloning vector is a DNA molecule that can carry foreign DNA into a host cell and replicate there © 2011 Pearson Education, Inc.
- 43. Producing Clones of Cells Carrying Recombinant Plasmids • Several steps are required to clone the hummingbird β-globin gene in a bacterial plasmid – The hummingbird genomic DNA and a bacterial plasmid are isolated – Both are cut with the same restriction enzyme – The fragments are mixed, and DNA ligase is added to bond the fragment sticky ends © 2011 Pearson Education, Inc.
- 44. © 2011 Pearson Education, Inc. Animation: Cloning a Gene Right-click slide / select “Play”
- 45. – Some recombinant plasmids now contain hummingbird DNA – The DNA mixture is added to bacteria that have been genetically engineered to accept it – The bacteria are plated on a type of agar that selects for the bacteria with recombinant plasmids – This results in the cloning of many hummingbird DNA fragments, including the β-globin gene © 2011 Pearson Education, Inc.
- 46. Figure 20.4 Bacterial plasmid TECHNIQUE RESULTS ampR gene lacZ gene Restriction site Hummingbird cell Sticky ends Gene of interest Humming- bird DNA fragments Recombinant plasmids Nonrecombinant plasmid Bacteria carrying plasmids Colony carrying non- recombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene One of many bacterial clones
- 47. Figure 20.4a-1 Bacterial plasmid TECHNIQUE ampR gene lacZ gene Restriction site Hummingbird cell Sticky ends Gene of interest Humming- bird DNA fragments
- 48. Figure 20.4a-2 Bacterial plasmid TECHNIQUE ampR gene lacZ gene Restriction site Hummingbird cell Sticky ends Gene of interest Humming- bird DNA fragments Recombinant plasmids Nonrecombinant plasmid
- 49. Figure 20.4a-3 Bacterial plasmid TECHNIQUE ampR gene lacZ gene Restriction site Hummingbird cell Sticky ends Gene of interest Humming- bird DNA fragments Recombinant plasmids Nonrecombinant plasmid Bacteria carrying plasmids
- 50. Figure 20.4b RESULTS Bacteria carrying plasmids Colony carrying non- recombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene One of many bacterial clones
- 51. Storing Cloned Genes in DNA Libraries • A genomic library that is made using bacteria is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome • A genomic library that is made using bacteriophages is stored as a collection of phage clones © 2011 Pearson Education, Inc.
- 52. Figure 20.5 Foreign genome Cut with restriction enzymes into either small fragments large fragments or Recombinant plasmids Plasmid clone (a) Plasmid library (b) BAC clone Bacterial artificial chromosome (BAC) Large insert with many genes (c) Storing genome libraries
- 53. Figure 20.5a (c) Storing genome libraries
- 54. • A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insert • BACs are another type of vector used in DNA library construction © 2011 Pearson Education, Inc.
- 55. • A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell • A cDNA library represents only part of the genome—only the subset of genes transcribed into mRNA in the original cells © 2011 Pearson Education, Inc.
- 56. Figure 20.6-1 DNA in nucleus mRNAs in cytoplasm
- 57. Figure 20.6-2 DNA in nucleus mRNAs in cytoplasm mRNA Reverse transcriptase Poly-A tail DNA strand Primer 5 5 3 3 A A A A A A T T T T T
- 58. Figure 20.6-3 DNA in nucleus mRNAs in cytoplasm mRNA Reverse transcriptase Poly-A tail DNA strand Primer 5 5 5 5 3 3 3 3 A A A A A A A A A A A A T T T T T T T T T T
- 59. Figure 20.6-4 DNA in nucleus mRNAs in cytoplasm mRNA Reverse transcriptase Poly-A tail DNA strand Primer DNA polymerase 5 5 5 5 5 5 3 3 3 3 3 3 A A A A A A A A A A A A T T T T T T T T T T
- 60. Figure 20.6-5 DNA in nucleus mRNAs in cytoplasm mRNA Reverse transcriptase Poly-A tail DNA strand Primer DNA polymerase cDNA 5 5 5 5 5 5 5 5 3 3 3 3 3 3 3 3 A A A A A A A A A A A A T T T T T T T T T T
- 61. Screening a Library for Clones Carrying a Gene of Interest • A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene • This process is called nucleic acid hybridization © 2011 Pearson Education, Inc.
- 62. • A probe can be synthesized that is complementary to the gene of interest • For example, if the desired gene is – Then we would synthesize this probe © 2011 Pearson Education, Inc. 5 3 CTCATCACCGGC 5 3 GAGTAGTGGCCG
- 63. • The DNA probe can be used to screen a large number of clones simultaneously for the gene of interest • Once identified, the clone carrying the gene of interest can be cultured © 2011 Pearson Education, Inc.
- 64. Figure 20.7 Radioactively labeled probe molecules Gene of interest Probe DNA Single- stranded DNA from cell Film Location of DNA with the complementary sequence Nylon membrane Nylon membrane Multiwell plates holding library clones TECHNIQUE 5 5 3 3 GAGTAGTGGCCG CTCATCACCGGC
- 65. Expressing Cloned Eukaryotic Genes • After a gene has been cloned, its protein product can be produced in larger amounts for research • Cloned genes can be expressed as protein in either bacterial or eukaryotic cells © 2011 Pearson Education, Inc.
- 66. Bacterial Expression Systems • Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells • To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active bacterial promoter © 2011 Pearson Education, Inc.
- 67. Eukaryotic Cloning and Expression Systems • Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells, such as yeasts, as hosts for cloning and expressing genes • Even yeasts may not possess the proteins required to modify expressed mammalian proteins properly • In such cases, cultured mammalian or insect cells may be used to express and study proteins © 2011 Pearson Education, Inc.
- 68. • One method of introducing recombinant DNA into eukaryotic cells is electroporation, applying a brief electrical pulse to create temporary holes in plasma membranes • Alternatively, scientists can inject DNA into cells using microscopically thin needles • Once inside the cell, the DNA is incorporated into the cell’s DNA by natural genetic recombination © 2011 Pearson Education, Inc.
- 69. Cross-Species Gene Expression and Evolutionary Ancestry • The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species • For example, Pax-6 is a gene that directs formation of a vertebrate eye; the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye) • The Pax-6 genes in flies and vertebrates can substitute for each other © 2011 Pearson Education, Inc.
- 70. Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) • The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA • A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules • The key to PCR is an unusual, heat-stable DNA polymerase called Taq polymerase. © 2011 Pearson Education, Inc.
- 71. Figure 20.8 Genomic DNA Target sequence Denaturation Annealing Extension Primers New nucleotides Cycle 1 yields 2 molecules Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence 5 5 5 5 3 3 3 3 2 3 1 TECHNIQUE
- 75. Figure 20.8d Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence
- 76. DNA technology allows us to study the sequence, expression, and function of a gene • DNA cloning allows researchers to – Compare genes and alleles between individuals – Locate gene expression in a body – Determine the role of a gene in an organism • Several techniques are used to analyze the DNA of genes © 2011 Pearson Education, Inc.
- 77. Gel Electrophoresis and Southern Blotting • One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis • This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size, electrical charge, and other properties • A current is applied that causes charged molecules to move through the gel • Molecules are sorted into “bands” by their size © 2011 Pearson Education, Inc.
- 78. © 2011 Pearson Education, Inc. Animation: Biotechnology Lab Right-click slide / select “Play”
- 79. Figure 20.9 Mixture of DNA mol- ecules of different sizes Power source Power source Longer molecules Cathode Anode Wells Gel Shorter molecules TECHNIQUE RESULTS 1 2
- 80. Figure 20.9a Mixture of DNA mol- ecules of different sizes Power source Power source Longer molecules Cathode Anode Wells Gel Shorter molecules TECHNIQUE 2 1
- 82. • In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis • Restriction fragment analysis can be used to compare two different DNA molecules, such as two alleles for a gene, if the nucleotide difference alters a restriction site © 2011 Pearson Education, Inc.
- 83. • Variations in DNA sequence are called polymorphisms • Sequence changes that alter restriction sites are called RFLPs (restriction fragment length polymorphisms) © 2011 Pearson Education, Inc.
- 84. Figure 20.10 Normal -globin allele Sickle-cell mutant -globin allele Large fragment Normal allele Sickle-cell allele 201 bp 175 bp 376 bp (a) DdeI restriction sites in normal and sickle-cell alleles of the -globin gene (b)Electrophoresis of restriction fragments from normal and sickle-cell alleles 201 bp 175 bp 376 bp Large fragment Large fragment DdeI DdeI DdeI DdeI DdeI DdeI DdeI
- 85. Figure 20.10a Normal -globin allele Sickle-cell mutant -globin allele (a) DdeI restriction sites in normal and sickle-cell alleles of the -globin gene 201 bp 175 bp 376 bp Large fragment Large fragment DdeI DdeI DdeI DdeI DdeI DdeI DdeI
- 86. Figure 20.10b Large fragment Normal allele Sickle-cell allele 201 bp 175 bp 376 bp (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles
- 87. • A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization • Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel © 2011 Pearson Education, Inc.
- 88. Figure 20.11 DNA restriction enzyme 3 2 1 4 TECHNIQUE I Normal -globin allele II Sickle-cell allele III Heterozygote Restriction fragments Nitrocellulose membrane (blot) Heavy weight Gel Sponge Alkaline solution Paper towels II I III II I III II I III Preparation of restriction fragments Gel electrophoresis DNA transfer (blotting) Radioactively labeled probe for -globin gene Nitrocellulose blot Probe base-pairs with fragments Fragment from sickle-cell -globin allele Fragment from normal - globin allele Film over blot Hybridization with labeled probe Probe detection 5
- 89. DNA Sequencing • Relatively short DNA fragments can be sequenced by the dideoxy chain termination method, the first automated method to be employed • Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths • Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment • The DNA sequence can be read from the resulting spectrogram © 2011 Pearson Education, Inc.
- 90. Figure 20.12 DNA (template strand) TECHNIQUE 5 3 C C C C T T T G G A A A A G T T T DNA polymerase Primer 5 3 P P P OH G dATP dCTP dTTP dGTP Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) P P P H G ddATP ddCTP ddTTP ddGTP 5 3 C C C C T T T G G A A A A DNA (template strand) Labeled strands Shortest Longest 5 3 ddC ddG ddA ddA ddA ddG ddG ddT ddC G T T T G T T T C G T T T C T T G G T T T C T G A G T T T C T G A A G T T T C T G A A G G T T T C T G A A G T G T T T C T G A A G T C G T T T C T G A A G T C A Direction of movement of strands Longest labeled strand Detector Laser Shortest labeled strand RESULTS Last nucleotide of longest labeled strand Last nucleotide of shortest labeled strand G G G A A A C C T
- 91. Figure 20.12a DNA (template strand) TECHNIQUE Primer Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) DNA polymerase 5 5 3 3 OH H G G dATP dCTP dTTP dGTP P P P P P P ddATP ddCTP ddTTP ddGTP T T T G G G C C C C T T T A A A A
- 92. Figure 20.12b DNA (template strand) Labeled strands Shortest Longest Direction of movement of strands Longest labeled strand Detector Laser Shortest labeled strand TECHNIQUE (continued) 5 3 G G C C C C T T T A A A A T T T G ddC ddC ddG ddG ddG ddA ddA ddA ddT 3 5 T T T G C T T T G C G T T T G C G A T T T G C G A A T T T G C G A A G T T T C G A A G T T T T C G A A G T C A T T T C G A A G T C G G G
- 93. Figure 20.12c RESULTS Last nucleotide of longest labeled strand Last nucleotide of shortest labeled strand G G G A A A C C T Direction of movement of strands Longest labeled strand Detector Laser Shortest labeled strand
- 94. Analyzing Gene Expression • Nucleic acid probes can hybridize with mRNAs transcribed from a gene • Probes can be used to identify where or when a gene is transcribed in an organism © 2011 Pearson Education, Inc.
- 95. Studying the Expression of Single Genes • Changes in the expression of a gene during embryonic development can be tested using – Northern blotting – Reverse transcriptase-polymerase chain reaction • Both methods are used to compare mRNA from different developmental stages © 2011 Pearson Education, Inc.
- 96. • Northern blotting combines gel electrophoresis of mRNA followed by hybridization with a probe on a membrane • Identification of mRNA at a particular developmental stage suggests protein function at that stage © 2011 Pearson Education, Inc.
- 97. • Reverse transcriptase-polymerase chain reaction (RT-PCR) is quicker and more sensitive because it requires less mRNA than Northern blotting • Reverse transcriptase is added to mRNA to make cDNA, which serves as a template for PCR amplification of the gene of interest • The products are run on a gel and the mRNA of interest is identified © 2011 Pearson Education, Inc.
- 98. Figure 20.13 cDNA synthesis PCR amplification Gel electrophoresis mRNAs cDNAs Primers -globin gene Embryonic stages 1 2 3 4 5 6 2 3 1 RESULTS TECHNIQUE
- 99. • In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism © 2011 Pearson Education, Inc.
- 100. Figure 20.14 50 m
- 101. Studying the Expression of Interacting Groups of Genes • Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays • DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions © 2011 Pearson Education, Inc.
- 102. Isolate mRNA. 2 1 3 4 TECHNIQUE Make cDNA by reverse transcription, using fluorescently labeled nucleotides. Apply the cDNA mixture to a microarray, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample. Tissue sample mRNA molecules Labeled cDNA molecules (single strands) DNA fragments representing a specific gene DNA microarray DNA microarray with 2,400 human genes Figure 20.15
- 103. Figure 20.15a DNA microarray with 2,400 human genes
- 104. Determining Gene Function • One way to determine function is to disable the gene and observe the consequences • Using in vitro mutagenesis, mutations are introduced into a cloned gene, altering or destroying its function • When the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotype © 2011 Pearson Education, Inc.
- 105. • Gene expression can also be silenced using RNA interference (RNAi) • Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the gene’s mRNA © 2011 Pearson Education, Inc.
- 106. • In humans, researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition • Genetic markers called SNPs (single nucleotide polymorphisms) occur on average every 100– 300 base pairs • SNPs can be detected by PCR, and any SNP shared by people affected with a disorder but not among unaffected people may pinpoint the location of the disease-causing gene © 2011 Pearson Education, Inc.
- 108. • Organismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell Cloning organisms may lead to production of stem cells for research and other applications © 2011 Pearson Education, Inc.
- 109. Cloning Plants: Single-Cell Cultures • One experimental approach for testing genomic equivalence is to see whether a differentiated cell can generate a whole organism • A totipotent cell is one that can generate a complete new organism • Plant cloning is used extensively in agriculture © 2011 Pearson Education, Inc.
- 110. Figure 20.17 Cross section of carrot root 2-mg fragments Fragments were cultured in nu- trient medium; stirring caused single cells to shear off into the liquid. Single cells free in suspension began to divide. Embryonic plant developed from a cultured single cell. Plantlet was cultured on agar medium. Later it was planted in soil. Adult plant
- 111. Cloning Animals: Nuclear Transplantation • In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell • Experiments with frog embryos have shown that a transplanted nucleus can often support normal development of the egg • However, the older the donor nucleus, the lower the percentage of normally developing tadpoles © 2011 Pearson Education, Inc.
- 112. Frog embryo Frog egg cell Frog tadpole UV Less differ- entiated cell Donor nucleus trans- planted Enucleated egg cell Fully differ- entiated (intestinal) cell Donor nucleus trans- planted Egg with donor nucleus activated to begin development Most develop into tadpoles. Most stop developing before tadpole stage. EXPERIMENT RESULTS Figure 20.18
- 113. Reproductive Cloning of Mammals • In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell • Dolly’s premature death in 2003, as well as her arthritis, led to speculation that her cells were not as healthy as those of a normal sheep, possibly reflecting incomplete reprogramming of the original transplanted nucleus © 2011 Pearson Education, Inc.
- 114. Figure 20.19 Mammary cell donor 2 1 3 4 5 6 TECHNIQUE RESULTS Cultured mammary cells Egg cell from ovary Egg cell donor Nucleus removed Cells fused Grown in culture Implanted in uterus of a third sheep Embryonic development Nucleus from mammary cell Early embryo Surrogate mother Lamb (“Dolly”) genetically identical to mammary cell donor
- 115. Figure 20.19a Mammary cell donor 2 1 3 TECHNIQUE Cultured mammary cells Egg cell from ovary Egg cell donor Nucleus removed Cells fused Nucleus from mammary cell
- 116. 4 5 6 RESULTS Grown in culture Implanted in uterus of a third sheep Embryonic development Nucleus from mammary cell Early embryo Surrogate mother Lamb (“Dolly”) genetically identical to mammary cell donor Figure 20.19b
- 117. • Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs • CC (for Carbon Copy) was the first cat cloned; however, CC differed somewhat from her female “parent” • Cloned animals do not always look or behave exactly the same © 2011 Pearson Education, Inc.
- 118. Figure 20.20
- 119. Problems Associated with Animal Cloning • In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth, and many cloned animals exhibit defects • Many epigenetic changes, such as acetylation of histones or methylation of DNA, must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development © 2011 Pearson Education, Inc.
- 120. Stem Cells of Animals • A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types • Stem cells isolated from early embryos at the blastocyst stage are called embryonic stem (ES) cells; these are able to differentiate into all cell types • The adult body also has stem cells, which replace nonreproducing specialized cells © 2011 Pearson Education, Inc.
- 121. Figure 20.21 Cultured stem cells Different culture conditions Different types of differentiated cells Embryonic stem cells Adult stem cells Cells generating all embryonic cell types Cells generating some cell types Liver cells Nerve cells Blood cells
- 122. • Researchers can transform skin cells into ES cells by using viruses to introduce stem cell master regulatory genes • These transformed cells are called iPS cells (induced pluripotent cells) • These cells can be used to treat some diseases and to replace nonfunctional tissues © 2011 Pearson Education, Inc.
- 123. Figure 20.22 Remove skin cells from patient. 2 1 3 4 Reprogram skin cells so the cells become induced pluripotent stem (iPS) cells. Patient with damaged heart tissue or other disease Return cells to patient, where they can repair damaged tissue. Treat iPS cells so that they differentiate into a specific cell type.
- 124. The practical applications of DNA technology affect our lives in many ways • Many fields benefit from DNA technology and genetic engineering © 2011 Pearson Education, Inc.
- 125. Medical Applications • One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases © 2011 Pearson Education, Inc.
- 126. Diagnosis and Treatment of Diseases • Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers, then sequencing the amplified product to look for the disease-causing mutation • SNPs may be associated with a disease-causing mutation • SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer © 2011 Pearson Education, Inc.
- 127. Human Gene Therapy • Gene therapy is the alteration of an afflicted individual’s genes • Gene therapy holds great potential for treating disorders traceable to a single defective gene • Vectors are used for delivery of genes into specific types of cells, for example bone marrow • Gene therapy provokes both technical and ethical questions © 2011 Pearson Education, Inc.
- 128. Figure 20.23 Cloned gene 2 1 3 4 Retrovirus capsid Bone marrow cell from patient Viral RNA Bone marrow Insert RNA version of normal allele into retrovirus. Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Viral DNA carrying the normal allele inserts into chromosome. Inject engineered cells into patient.
- 129. Pharmaceutical Products • Advances in DNA technology and genetic research are important to the development of new drugs to treat diseases © 2011 Pearson Education, Inc.
- 130. • The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor • Pharmaceutical products that are proteins can be synthesized on a large scale Synthesis of Small Molecules for Use as Drugs © 2011 Pearson Education, Inc.
- 131. • Host cells in culture can be engineered to secrete a protein as it is made, simplifying the task of purifying it • This is useful for the production of insulin, human growth hormones, and vaccines Protein Production in Cell Cultures © 2011 Pearson Education, Inc.
- 132. • Transgenic animals are made by introducing genes from one species into the genome of another animal • Transgenic animals are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use Protein Production by “Pharm” Animals © 2011 Pearson Education, Inc.
- 133. Figure 20.24
- 134. Figure 20.24a
- 135. Figure 20.24b
- 136. Forensic Evidence and Genetic Profiles • An individual’s unique DNA sequence, or genetic profile, can be obtained by analysis of tissue or body fluids • DNA testing can identify individuals with a high degree of certainty • Genetic profiles can be analyzed using RFLP analysis by Southern blotting © 2011 Pearson Education, Inc.
- 137. • Even more sensitive is the use of genetic markers called short tandem repeats (STRs), which are variations in the number of repeats of specific DNA sequences • PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths • The probability that two people who are not identical twins have the same STR markers is exceptionally small © 2011 Pearson Education, Inc.
- 138. Figure 20.25 This photo shows Washington just before his release in 2001, after 17 years in prison. (a) (b)These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder. Semen on victim Earl Washington Kenneth Tinsley 17,19 16,18 17,19 13,16 14,15 13,16 12,12 11,12 12,12 Source of sample STR marker 1 STR marker 2 STR marker 3
- 139. Figure 20.25a This photo shows Washington just before his release in 2001, after 17 years in prison. (a)
- 140. Environmental Cleanup • Genetic engineering can be used to modify the metabolism of microorganisms • Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials © 2011 Pearson Education, Inc.
- 141. Agricultural Applications • DNA technology is being used to improve agricultural productivity and food quality • Genetic engineering of transgenic animals speeds up the selective breeding process • Beneficial genes can be transferred between varieties or species © 2011 Pearson Education, Inc.
- 142. • Agricultural scientists have endowed a number of crop plants with genes for desirable traits • The Ti plasmid is the most commonly used vector for introducing new genes into plant cells • Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance, increased resistance to pests, increased resistance to salinity, and improved nutritional value of crops © 2011 Pearson Education, Inc.
- 143. Figure 20.26 Plant with new trait RESULTS TECHNIQUE Ti plasmid Site where restriction enzyme cuts DNA with the gene of interest Recombinant Ti plasmid T DNA Agrobacterium tumefaciens
- 144. Safety and Ethical Questions Raised by DNA Technology • Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures • Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology © 2011 Pearson Education, Inc.
- 145. • Most public concern about possible hazards centers on genetically modified (GM) organisms used as food • Some are concerned about the creation of “super weeds” from the transfer of genes from GM crops to their wild relatives • Other worries include the possibility that transgenic protein products might cause allergic reactions © 2011 Pearson Education, Inc.
- 146. • As biotechnology continues to change, so does its use in agriculture, industry, and medicine • National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology © 2011 Pearson Education, Inc.
- 148. Figure 20.UN04 DNA fragments from genomic DNA or cDNA or copy of DNA obtained by PCR Cloning vector Mix and ligate Recombinant DNA plasmids
- 149. Figure 20.UN05 Aardvark DNA Plasmid 5 3 5 3 TCCATGAATTCTAAAGCGCTTATGAATTCACGGC AGGTACTTAAGATTTCGCGAATACTTAAGTGCCG
- 150. Figure 20.UN06
- 151. Figure 20.UN07
- 152. Figure 20.UN08