DNA Technologies and Genomics
Chapter 18
Why It Matters
Snowball: Key to a Murder
Biotechnology
Biotechnology• Any technique applied to biological systems to
manipulate processes
DNA technologies isolate purify, analyze and manipulate DNA sequences• DNA fingerprinting used in forensics
Genetic engineering uses DNA technologies to alter genes for practical purposes
18.1 DNA Cloning
Bacterial enzymes (restriction endonucleases) form the basis of DNA cloning
Bacterial plasmids illustrate the use of restriction enzymes in cloning
DNA libraries contain collections of cloned DNA fragments
Polymerase chain reaction (PCR) amplifies DNA in vitro
Recombinant DNA
DNA cloning provides many copies of a gene• Used for research or manipulation
Recombinant DNA contains DNA from multiple sources joined together• Recombinant plasmids containing gene of
interest can be cloned in E. coli
Cloning DNA Fragments
Endonucleases
Restriction enzymes (endunucleases) cut DNA at specific sequences in restriction sites• Restriction fragments result • Sticky ends have unpaired bases at cuts which
will hydrogen bond• Ligase stitches together paired sticky ends
Restriction Enzyme EcoRI
Plasmid Cloning Vectors
Engineered to contain gene of interest and sorting genes• Sorting genes identify E. coli with cloned plasmid• E. coli with appropriate plasmid are ampicillin
resistant and blue-white screened on X-gal
Plasmid Cloning
DNA Hybridization
Uses nucleic acid probe to identify gene of interest in set of clones• Probe has tag for detection• Identified colony produces large quantities of
cloned gene
DNA Hybridization
DNA Libraries
Genomic libary • Clones containing every sequence in a genome• Used to isolate genes or DNA sequences
Complementary DNA (cDNA) library • DNA sequences made from expressed RNA• mRNA extracted from cell• Reverse transcriptase makes cDNA• Removes introns for genetic engineering
Polymerase Chain Reaction
Polymerase chain reaction (PCR) • Produces many sequence copies without host
cloning• Amplifies known DNA sequences for analysis• Only copies sequence of interest • Primers bracket sequence
Agarose gel electrophoresis • Separates fragments by size and charge• Gel molecular sieve
Polymerase Chain Reaction
Agarose Gel Electrophoresis
18.2 Application of DNA Technologies
DNA technologies are used in molecular testing for many human genetic diseases
DNA fingerprinting used to identify human individuals and individuals of other species
Genetic engineering uses DNA technologies to alter the genes of a cell or organism
DNA technologies and genetic engineering are a subject of public concern
RFLPs
Restriction fragment length polymorphisms• DNA sequence length changes due to varying
restriction sites from same region of genome• Sickle cell anemia has RFLPs
Southern blot analysis uses electrophoresis, blot transfer, and labeled probes to identify RFLPs• Alternative is PCR and electrophoresis
Sickle-Cell RFLPs
Southern Blot Analysis
DNA Fingerprinting
Distinguishes between individuals• Uses PCR at multiple loci within genome• Each locus heterozygous or homzygous for short
tandem repeats (STR)
PCR amplifies DNA from STR• Number of gel electrophoresis bands shows
amplified STR alleles• 13 loci commonly used in human DNA
fingerprinting
Forensics and Ancestry
Forensics compares DNA fingerprint from sample to suspect or victim• Usually reported as probability DNA came from
random individual
Common alleles between children and parents used in paternity tests• Same principle used to determine evolutionary
relationships between species
DNA Fingerprint
Genetic Engineering
Transgenic organisms • Modified to contain genes from external source
Expression vector has promoter in plasmid for production of transgenic proteins in E. coli• Example: Insulin• Protocols to reduce risk of escape
Animal Genetic Engineering
Transgenic animals used in research, correcting genetic disorders, and protein production
Germ-line cell transgenes can be passed to offspring (somatic can not)• Embryonic germ-line cells cultured in quantity,
made into sperm or eggs• Stem cells
Transgenic Mice
Genetically Engineered Mouse
Gene Therapy
Attempts to correct genetic disorders• Germ-line gene therapy can’t be used on humans• Somatic gene therapy used in humans
Mixed results in humans• Successes for ADA and sickle-cell• Deaths from immune response and leukemia-like
conditions
Animal Genetic Engineering
“Pharm” animals produce proteins for humans • Usually produced in milk for harmless extraction
Cloned mammals produced by implantation of diploid cell fused with denucleated egg cell• Low cloning success rate• Increased health defects in clones• Gene expression regulation abnormal
Cloned Sheep
“Dolly”
Plant Genetic Engineering
Has been highly successful• Increased resistance to environmental effects
and pathogens• Plant “pharms” and increased nutrition• Callus formation
Ti (tumor inducing) plasmid from crown gall disease used as vector• Transforming DNA (T DNA) genes expressed
Crown Gall Tumor
Ti Plasmid and Transgenic Plants
Fig. 18-15b, p. 389
Plant cell (not to scale)
Nucleus
Regeneratedtransgenicplant
T DNA with gene ofinterest integrated intoplant cell chromosome
Rhizobium radiobacterdisarmed so cannotinduce tumors
GMO Concerns
Genetically modifed organisms (GMOs) are transgenic and raise certain concerns• Effect on environment• Interbreeding with or harming natural species
Cartagena Protocol on Biosafety provides rules on GMOs • Stringent laboratory standards for transgenic
organisms• No bacterial “escapes” from labs
GMO Tobacco
GMO Rice
18.3 Genome Analysis
DNA sequencing techniques are based on DNA replication
Structural genomics determines the complete DNA sequence of genomes
Functional genomics focuses on the functions of genes and other parts of the genome
18.3 (cont.)
Studying the array of expressed proteins is the next level of genomic analysis
Systems biology is the study of the interactions between all the components of an organism
Genome Analysis
Genomics • Analyzes organization of complete genome and
gene networks
Human Genome Project took 13 years (2003)• Revolutionizing biology and evolutionary
understanding
DNA Sequencing
Used for small DNA sequences to genomes
Dideoxy (Sanger) method of sequencing• Dideoxyribonucleotides have –H bound to 3’ C
instead of –OH
• DNA polymerases place dideoxyribonucleotides in DNA, stops replication
• Polyacrylamide gel separates strands varying by one nucleotide
Dideoxy (Sanger) Method
Genomic Analyses (1)
Structural genomics • Sequence genomes to locate genes and funtional
sequenes
Functional genomics • Studies functions of genes and other parts of
genome
Genomic Analyses (2)
Whole-genome shotgun method • Breaks genome into many DNA fragments
• Computers assemble genome based on overlapping sequences
Whole-Genome Shotgun Sequencing
Functional Genomics
Bioinformatics • Analysis of large data sets
• Uses biology, computer science, mathematics
• Identify open reading frames with start and stop codons, sophisticated algorithms for introns
• Sequence similarity searches
Genomics revealed many unknown genes• Many genes similar between evolutionarily distant
organisms
Human Genome
3.2 billion base pairs
Between 20,000 and 25,000 genes
About 100,000 proteins• Due to alternative splicing and protein processing
Protein coding only 2% of genome• 24% introns
• 50% repeat sequences of no known function
Genome Analysis
Data mining• Gene functions
• Genome organization
• Expression controls
Comparative genomics (with other organisms)• Tests evolutionary hypotheses
DNA Microarrays
DNA microarrays (chips) • About 20 nucleotide-long DNA probe sequences
• cDNA probes made from isolated mRNA
• Probes red or green from different cell states
• cDNA from each cell state hybridize with complementary sequences on chip
Used to determine how expression changes in normal and cancer cells• Also used to detect mutations
DNA Microarray Analysis
Proteomics
Proteome • Complete set of proteins expressed by genome
• Larger than genome in eukaryotes
Proteomics (study of proteome)• Protein microarrays (chips) similar to DNA
microarrays
• Use antibodies to bind to proteins
Systems Biology
Studies organisms as a whole• Investigates networks of genes, proteins, and
biochemistry
Combines genomics and proteomics with response to environment• Complex data analysis and computer models
limiting factors