DNA TechnologyProblem:A chromosome can be millions of base pairs long.How do you isolate and study a single gene?Scientists had to find out how to cut, paste, and copy DNA.

Fragile X chromosome

Cutting and Pasting DNA• 1962 – DNA cutting enzymes isolated from

E. coli = restriction endonucleases, aka “molecular scissors”

- left “sticky ends”

• Arthur Kornberg identified the pasting

mechanism for DNA- an enzyme called ligase- made artificial viral DNA loops

Werner Arber

Arthur KornbergMade “life in a

test tube”

Restriction Enzymes

Gene CloningCurrent applications:A gene that originated in one organism can be used to giveanother organism a new metabolic capability.

Useful protein products can beharvested in large quantities from bacterial cultures.

Can also be used for basic research on genes or proteins.

Molecular cloning – isolating a defined sequence of DNA and making multiple copies of it “in vivo”.

Using Reverse Transcriptase to Synthesize

cDNA• Reverse transcriptase – an RNA-dependent DNA polymerase

• Encoded by retroviruses – copy viral RNA into DNA prior to integration into host

•Transcribes both ss RNA and ss DNA (needs a primer)•Degrades the RNA from RNA-DNA hybrids.

•Used to copy RNA into complementary DNAs (cDNAs).

Making cDNA for a Eukaryotic Gene Using Reverse

Transcriptase

Cloning a Eukaryotic Gene in a Bacterial Plasmid

X-gal is hydrolyzed by B-galactosidaseto yield a blue product.If a plasmid has foreign DNA insertedinto its lac Z gene, then the colony ofcells producing it will be white.

Can synthesize a nucleic acid probe -cDNA (complementary to the gene of interest) labeled with a radioactive isotope or a fluorescent tag.

A “shotgun” approach

Using a Nucleic Acid Probe to Identify a Cloned Gene

Cloning a eukaryotic gene into a prokaryotic cell: problems and

solutions• Differences in gene expression (ex: promoters and other

DNA control sequences).- Solution: expression vector - contains a prokaryotic promoter just before the site where the eukaryotic gene is inserted.

• Presence of long introns in most eukaryotic genes (bacterial cells do not have RNA-splicing machinery).

- Solution: make artificial eukaryotic genes that lack introns. -extract processed RNA from the eukaryotic nucleus (no

introns)-use reverse transcriptase to make cDNA transcripts of this

RNA.-cDNA is attached to vector DNA for replication inside a cell.-vector provides a bacterial promoter and any other

necessary control elements

Using Eukaryotic Cells for Cloning: Yeast

• Avoids eukaryotic-prokaryotic incompatibility• Yeast – single-celled, easy to grow, have plasmids• Scientists have constructed vectors called

yeast artificial chromosomes (YACs) -have an origin for DNA replication, a centromere, and two telomeres-behave normally in mitosis – foreign DNA cloned as the yeast cell divides-can carry more DNA than a plasmid vector

• Problem: many eukaryotic proteins have to be modified before they are functional (addition of carbohydrate or lipid groups)

-Yeast cells may not be able to modify the protein correctly

Artificial Chromosomes

Mammalian satellite DNA-basedartificial chromosomes (SATACs)

Use of host cells from animal or plant cell cultures

• Many kinds of eukaryotic cells can take up foreign DNA – but not very efficiently

• Scientists have developed a variety of more aggressive methods:

-electroporation – apply a brief electrical pulse to a solution

containing cells. Creates a temporary hole in the CM thru which DNA can enter.-inject DNA directly into cells with a microscopic needle-in plants, can attach DNA to microscopic particles of metals and fire the particles into cells with a “gene gun”

DNA Libraries• DNA fragments cloned at random into a plasmid

vector - the majority of genetic information will be included in the mixture of bacteria (“shotgun” approach)

• Cultures of the bacteria, collectively contain all the genes and are called a library.

cDNA Libraries

• Partial genomic library• Produced using the mRNA molecules

isolated from a cell.• Contains only the genes that are

expressed (transcribed) within the cell.

• Advantage – can study the genes responsible for specialized functions in specific cell types.

The Polymerase Chain Reaction (PCR)

• A technique of quickly amplifying DNA without using cells

• DNA contains the sequence “targeted” for copying

• A heat-resistant DNA polymerase is added (isolated from bacteria living in hot springs!)

• Plus a supply of all four nucleotides and primers

• Primers are short, synthetic molecules of single-stranded DNA complementary to the ends of the targeted DNA

• Each cycle takes only about 5 minutes to complete

DNA Analysis and Genomics• Genomics – sequencing and studying whole sets of genes and

their interactions - to make comparisons between cells, individuals, and species.

• Gel electrophoresis – separates nucleic acids and proteins based on size and charge

• Pure samples of these bands can be recovered from the gel and retain their biological activity.

Restriction Fragment Analysis by Southern

Blotting1. Restriction enzyme applied.2. Gel separation.3. Blot onto nitrocellulose paper.4. Ss-DNA probes added.5. Hybridization.6. Bands identified ARG.

RFLP

Restriction Fragment Length Polymorphisms

(RFLPs)• Used to find differences in noncoding

sequences of DNA.

• Can serve as genetic markers for a particular location (locus) in the genome.

• A given RFLP marker frequently occurs in numerous variants in a population (hence, polymorphisms)

A DNA fingerprint is a specific pattern of RFLP

bands

STRs – variations innumber of tandem repeated base sequences found in satellite DNA.

Human DNA Fingerprinting

Father and four children.Which lane contains the DNA of the father?

Which child is least likely to be the biological offspring of these parents?

Lane 3 is the only lane that shares one band with each of the other lanes.

Child 2

The Human Genome Project

• Goals:1. Determine nucleotide sequence for entire human genome (3 X 109 bps).

2. Map the location of every gene on each chromosome.3. Compare to genomes of other organisms.

• Fundamental questions:1. How are genomes organized?2. How is gene expression controlled?3. How are cell growth and differentiation under genetic control?4. How does evolution occur?

• Scientists have discovered specific genes responsible for several genetic disorders:

1. Cystic fibrosis2. Duchenne muscular dystrophy3. Colon cancer

Human Genome Project• International consortium of 20 groups of researchers. • Included mapping of important research organisms –

E. coli, yeast, C.elegans (nematode), and mouse.• Three stages:

1. Genetic (linkage) mapping – develop a map of several thousand genetic markers (genes, RFLPs, STRs)2. Physical mapping: ordering DNA fragments using restriction fragments (overlap)3. DNA sequencing using clones of short DNA fragments and,

later, DNA sequencing machines.

• Later, J.C. Ventra (Celera Genomics) used powerful computer programs to order the large number of random short sequences cut from the whole genome.

Studying Gene Expression

DNA Microarray Assays

Red – genes expressed in sample A.Green – genes expressed insample B.Yellow – genes expressed equally in both samples.

Used to determine which genesare expressed in response to aspecific treatment or disease.Also – tissue specific genes.

DNA microarray - full set of 1,000sof sequences

Determining Gene Function

• In vitro mutagenesis:

1. Changes are made to a gene2. Altered gene returned to cell3. Monitor changes in physiology

or developmental patterns

• RNA interference (RNAi):

- Synthetic double-stranded RNA molecules that match a gene sequence trigger the breakdown of that gene’s mRNA.

Practical Applications of DNA Technology

• Medicine:-Diagnosis of infectious diseases and genetic disorders

-Human gene therapy – currently aimed at fighting heart disease and cancer-Pharmaceutical products – growth hormones, insulin, vaccines

• Forensics:-DNA fingerprints

-Use simple tandom repeats (STRs) found in satellite DNA-Five small regions of the genome known to vary widely

Practical Applications of DNA Technology (cont.)

• Environmental – genetically engineered organisms:– Able to extract heavy metals– Sewage treatment– Research – engineering organisms to degrade chlorinated

hydrocarbons and other toxic chemicals- Bioremediation – cleaning up oil spills and waste dumps

• Agricultural: - Transgenic organisms

-increased productivity-pest resistance, disease resistance-”pharm” animals-”golden rice” – enriched with beta-carotene

Safety and Ethical Questions