Chapter 20 DNA Technology & Genomics. If we can, should we?

Chapter 20 DNA Technology & Genomics If we can, should we?

Biotechnology Genetic manipulation of organisms or their components to make useful products Humans have been doing this for 1,000s of years plant & animal breeding

Biotechnology Today Genetic Engineering Direct manipulation of DNA for practical purposes Why do this? diagnose diseases or defects cure diseases or defects change/improve organisms

Gene Cloning Why clone genes? 1. to produce a protein product 2. to prepare many copies of the gene itself

Recombinant DNA Contains genes from 2 different sources Made with restriction enzymes Enzymes made by bacteria that cut DNA at specific locations (why?) hundreds of different enzymes (EcoRI, HindIII, BamHI, SmaI) cuts at a restriction site specific sequence of DNA symmetrical palindrome produces sticky ends

Genes can be cloned in vectors called plasmids Plasmids small, self-replicating circular DNA molecules naturally occur in bacteria insert DNA sequence (gene of interest) into plasmid Transformation insert recombinant plasmid into bacteria culture (grow) recombinant bacteria = cell clones permits production of multiple copies of a specific gene or DNA sequence (gene clones) Why use bacteria?

Cloning a Human Gene using a bacterial plasmid 5 Steps: 1. Isolation of vector and gene-source DNA 2. Insertion of DNA into the vector Other possibilities?

Cloning a Human Gene 3. Introduction of the cloning vector into cells (transformation)

Cloning a Human Gene 4. Cloning of cells (and foreign genes) Use both ampicillin resistance & color to identify clones containing recombinant plasmids: only clones containing plasmids will survive ampicillin antibiotic on plate only clones with disrupted lacz gene (& foreign DNA) will be white (functional gene turns X-gal blue) 5. Identification of cell clones carrying the gene of interest (next slide )

Using a nucleic acid probe to identify a cloned gene of interest Probe short, single stranded DNA molecule mix with denatured DNA (single stranded) DNA Hybridization probe bonds to complementary DNA sequence (gene of interest) Label probe is labeled for easy detection (radioactivity or fluorescence)

But bacteria do it differently Eukaryotic gene In a bacterial cell see any problems? non-recognizable euk. promoter no RNA processing Solutions: Expression vectors (use prokaryotic promoters) cdna (gene lacking introns) mature mrna DNA (using reverse transcriptase) Yeast Artificial Chromosomes (YACs)

Essay Qs DNA Sequencing DNA to Protein (Transcription & Translation) Cloning a Gene DNA Fingerprinting

Genomic Libraries store cloned genes Can store an entire genome in a genomic library (1,000s of cloned genes)

Polymerase Chain Reaction Clones DNA in vitro Any piece of DNA can be copied many times WITHOUT using cells Need: Sequence of DNA to be copied DNA primers (crucial) Heat-resistant DNA polymerase Nucleotides (G, A, C, T)

II. DNA Analysis & Genomics So we ve got the DNA, now what?

Gel Electrophoresis works on nucleic acids (DNA/RNA) and proteins separates fragments by size, electrical charge, etc. (different rates of movement through a gel in an electric field)

restriction fragment patterns distinguish between DNA Differences in DNA sequences = different cuts made by restriction enzymes = different bands on the electrophoresis gel Can detect subtle differences between DNA sequences/alleles

Restriction Fragment Length Polymorphisms (RFLPs) Differences in non-coding DNA sequences that can result in different patterns of restriction fragment lengths Detected/analyzed with Southern blotting Useful as genetic markers for making linkage maps

Southern Blotting (restriction fragment analysis) Used to compare the DNA of different individuals or species Can look at differences in specific genes (using DNA probes)

Human Genome Project Three stages: Genetic (Linkage) Mapping Physical Mapping DNA Sequencing 3.2 billion base pairs

DNA Sequencing (Sanger method)

Strategies for Genome Sequencing

Physical Mapping Chromosome Walking

Essay Qs DNA Sequencing DNA to Protein (Transcription & Translation) Cloning a Gene DNA Fingerprinting

Studying Gene Expression DNA Microarrays (DNA Chips) tell us Which genes are active and when

1. The principal problem with inserting an unmodified mammalian gene into the bacterial chromosome, and then getting that gene expressed, is that a) prokaryotes use a different genetic code from that of eukaryotes. b) bacteria translate polycistronic messages only. c) bacteria cannot remove eukaryotic introns. d) bacterial RNA polymerase cannot make RNA complementary to mammalian DNA. e) bacterial DNA is not found in a membrane-enclosed nucleus and is therefore incompatible with mammalian DNA. Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

2. Which of the following statements is consistent with the results below? * a) B is the child of A and C. b) C is the child of A and B. c) D is the child of B and C. d) A is the child of B and C. e) A is the child of C and D. Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

3. Which of the following statements is most likely true? a) D is the child of A and C. b) D is the child of A and B. c) D is the child of B and C. d) A is the child of C and D. e) B is the child of A and C. Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

5. The segment of DNA shown in the figure below has restriction sites I and II, which create restriction fragments A, B, and C. Which of the gels produced by electrophoresis shown below would represent the separation and identity of these fragments? Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

6. This restriction fragment contains a gene whose recessive allele is lethal. The normal allele has restriction sites for the restriction enzyme PSTI at sites I and II. The recessive allele lacks restriction site I. An individual who had a sister with the lethal trait is being tested to determine if he is a carrier of that allele. Indicate which of these band patterns would be produced on a gel if he is a carrier (heterozygous for the gene)? Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings