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