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 b, p. 389 Plant cell (not to scale) Nucleus Regenerated transgenic plant T DNA with gene of interest integrated into plant cell chromosome Rhizobium radiobacter disarmed so cannot induce 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