A. Chapter 20~DNA

Technology &

Genomics

I. Intro: VocabularyA. Recombinant DNA: DNA in

which genes from 2 different sources are combined

B. Genetic engineering: direct manipulation of genes for practical purposes

C. Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products

Overview: Use of Bacterial Plasmids in Gene Cloning

II. DNA Cloning

A. Cloned genes used for basic research and commercial products

1. A foreign gene is inserted into a bacterial plasmid and this recombinant DNA molecule is returned to a bacterial cell.

2. Every time this cell reproduces, the recombinant plasmid is replicated as well and passed on to its descendents.

3. Under suitable conditions, the bacterial clone will make the protein encoded by the foreign gene.

B. Restriction enzymes (endonucleases): in nature, these enzymes protect bacteria from intruding DNA

1. they cut up the DNA (restriction)

2. very specific

C. Restriction site:

recognition sequence for a particular restriction enzyme

D. Restriction fragments: segments of DNA cut by restriction enzymes in a reproducable way

E. Sticky end: cut covalent phosphodiester bonds of both strands = short extensions of restriction fragments

F. DNA ligase: enzyme that can join the sticky ends of DNA fragments

G. Cloning vector: DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids)

III. Genes can be cloned in DNA vectors

A. Recombinant plasmids- splicing restriction fragments from foreign DNA into plasmid

1. can be returned relatively easily to bacteria2. cloning vector- a DNA molecule that can carry

foreign DNA into a cell and replicate there

B. As a bacterium carrying a recombinant plasmid reproduces, the plasmid replicates within it

C. Bacteria are most commonly used as host cells:

1. DNA can be easily isolated 2. Then, reintroduced into their cells3. the cultures grow (replicate) quickly

D. Steps for eukaryotic gene cloning

1. Isolation of cloning vector (bacterial plasmid) & gene-source DNA (gene of interest)

2. Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase

3. Introduction of cloning vector into cells (transformation by bacterial cells)

4. Cloning of cells (and foreign genes)5. Identification of cell clones carrying the

gene of interest, one way is nucleic acid hybridization using a nucleic acid probe

How do we know which colonies contain the cloned gene?

-Look for the gene or its protein product

Plasmid Cloning

http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/plasmidcloning_fla.html

– After denaturation (separating) the DNA strands in the plasmid, the probe will hydrogen-bond to its complementary sequence, tagging colonies with the targeted gene

F. Solutions to Problems Expressing Eukaryotic Genes

1. Use expression vector, a cloning vector containing the prokaryotic promotor upstream of the restriction site

1. bacterial host recognizes the promotor and expresses the foreign gene

2. Introns in eukaryotic genes1. A processed mRNA acts as the template for

making a complementary DNA (cDNA) by reverse transcription. cDNA, with a promoter, can be attached to a vector for replication, transcription, and translation inside bacteria.

cDNA

3. Use eukaryotic cells as host for genes1. Yeast cells, single-celled fungi, are as easy to

grow as bacteria and have plasmids, (rare for eukaryotes)

2. Scientists have constructed yeast artificial chromosomes (YACs) - an origin site for replication, a centromere, and two telomeres

3. carry more DNA than a plasmid 4. Host provides the modifications after

translation needed by many proteinsA. includes adding carbohydrates or lipids

4. When DNA not taken up efficiently1. electroporation, brief electrical pulses create

a temporary hole in plasma membrane2. Or, scientists can inject DNA into cells using

microscopically thin needle

G. Polymerase chain reaction (PCR)

1. Devised in 19852. Quick amplification of any piece of

DNA without cells (in vitro)3. PCR can make billions of copies of a

targeted DNA segment in a few hours

A. three-step cycle: heating, cooling, and replication

B. Applications: fossils (40,000 yr old wooly mammoth), forensics, prenatal diagnosis, etc.

http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html

DNA incubated in test tube w/:

-special DNA polymerase

-supply of nucleotides

-short pieces of DNA primer

IV. DNA Analysis & Genomics

A. Gel electrophoresisB. Restriction fragment

analysis (RFLPs)C. Southern blottingD. DNA sequencing

E. Human genome project

We have gene segments, now what?Genomics- Comparisons among whole sets of genes & interactions

A. Gel ElectropheresisA. Gel electrophoresis: separates nucleic

acids or proteins on the basis of size or electrical charge creating DNA bands of the same length

1. DNA molecule separation depends mainly on size (length of fragment) with longer fragments migrating less along the gel

Gel Electropheresis

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B. Restriction fragment analysis

1. Separated fragments can be recovered undamaged from gels, providing pure samples of individual fragments.

Distinguish different alleles (specific to one base pair)

2. Although electrophoresis will yield too many bands to distinguish individually, we can use nucleic acid hybridization with a specific probe to label discrete bands that derive from our gene of interest.

3. The radioactive label on the single-stranded probe can be detected by autoradiography

4. restriction fragment length polymorphisms (RFLPs) can serve as genetic markers for a particular location (locus) in the genome

C. Southern Blotting1. (Southern hybridization) the transfer

of the DNA fragments from the gel to a sheet of nitrocellulose paper

A. Fragments separated by sizeB. denatures the DNA fragments

2. Bathe sheet in solution containing a probe

A. probe attaches by base-pairing (hybridize) to the DNA sequence of interest

3. Visualize bands containing the label with autoradiography

Three individuals, the results of these steps show that individual III has a different restriction pattern than individuals I or II.

D. Entire genomes can be mapped at the DNA level

1. Human Genome Project, begun in 1990

A. RFLPs serve as the basis of a detailed map of the entire human genome

2. Other organisms important to biological research with entire genomes mapped: E. coli, yeast, fruit fly, and mouse

3. Three phases to sequencing: 1. genetic (linkage) mapping2. physical mapping3. DNA sequencing

4. Genetic mapping- use linkage maps to locate genetic markers throughout chromosomes

1. Based on recombination frequencies2. Markers may be known segements

of DNA, RFLPs, and microsatellites

5. Physical mapping- determining order of identified restriction fragments

1. Chromosome walking- using known segments to make a map of overlapping fragments

Chromosome walking

6. DNA sequencing- the long fragments are then cut, cloned and sequenced

7. Sanger Method- deriving the sequence in a method similar to PCR

1. Special dideoxynucleotides used in reaction, do not copy the whole template, instead, fragments of various lengths

2. dideoxynucleotides, marked radioactively or fluorescently, lack a 3’-OH to attach the next nucleotide

8. The order of these fragments via gel electrophoresis can be interpreted as the nucleotide sequence

J. Craig Venter (Celera Genomics) decided in 1992 to try a whole-genome shotgun approach

9. The progessA. In 1995, Venter announced genome of a

bacteriumB. In 2000, he finished Drosophila

melanogasterC. In February, 2001, Celera and the public

consortium separately announced sequencing over 90% of the human genome

D. By mid-2001, the genomes of about 50 species had been completely (or almost completely) sequenced

E. There are still gaps in the human sequence

enormous amounts of noncoding DNA

E. Evolutionary Significance1. Comparisons of genome sequences

confirm very strongly the evolutionary connections between even distantly related organisms and the relevance of research on simpler organisms to our understanding of human biology.

A. yeast genes can substitute for human versions B. Understand human disease gene by studying

its normal counterpart in yeastC. Bacterial sequences reveal unsuspected

metabolic pathways that may have industrial or medical uses

F. Unknown gene functions

1. disable the gene and hope that the consequences provide clues to the gene’s normal function

A. Using in vitro mutagenesis, specific changes are introduced into a cloned gene, altering or destroying its function.

B. When the mutated gene is returned to the cell, it may be possible to determine the function of the normal gene by examining the phenotype of the mutant.

2. In nonmammalian organisms, a simpler and faster method, RNA interference (RNAi), has been applied to silence the expression of selected genes.

What’s Next?A. The next step is proteomics, the

systematic study of full protein sets (proteomes) encoded by genomes.

B. Challenges:1. The sheer number of proteins in humans

due to:A. alternative RNA splicingB. post-translational modifications

2. Collecting proteins because a cell’s proteins differ with cell type and its state

3. Proteins are extremely varied in structure and chemical and physical properties

V. Practical DNA Technology Uses

A. Diagnosis of diseaseB. Human gene therapyC. Pharmaceutical products

(vaccines)D. ForensicsE. Animal husbandry (transgenic

organisms)F. Genetic engineering in plantsG. Ethical concerns?

Human Gene Therapy

DNA fingerprints can be used forensically to presence evidence to juries in murder trials.the blood on the clothes is from the victim,

not the defendant.

Agricultural Use

A. Crop plants with genes for desirable traits1. delayed ripening and resistance to spoilage

and disease2. Because a single transgenic plant cell can be

grown in culture to generate an adult plant, plants are easier to engineer than most animals

B. The Ti plasmid, from the soil bacterium Agrobacterium tumefaciens, is often used to introduce new genes into plant cells.

1. The Ti plasmid normally integrates a segment of its DNA into its host plant and induces tumors.

C.Foreign genes can be inserted into the Ti plasmid using recombinant DNA techniques-recombinant plasmid put back into

Agrobacterium, which then infects plant cells, or introduced directly into plant cells, only used in dicots (two seed leaves)