Understanding and Manipulating GenomesUnderstanding and Manipulating Genomes One of the greatest achievements of modern One of the greatest achievements of modern

sciencescience– Has been the sequencing of the human Has been the sequencing of the human

genome, which was largely completed by 2003genome, which was largely completed by 2003 DNA sequencing accomplishmentsDNA sequencing accomplishments

– Have all depended on advances in DNA Have all depended on advances in DNA technology, starting with the invention of technology, starting with the invention of methods for making methods for making recombinant DNArecombinant DNA

Chapter 20 & 21~Chapter 20 & 21~Biotechnology & GenomicsBiotechnology & Genomics

DNA technology has launched a DNA technology has launched a revolution in the area of revolution in the area of biotechnologybiotechnology– The manipulation of The manipulation of

organisms or their genetic organisms or their genetic components to make useful components to make useful productsproducts

Variation: Differences within Variation: Differences within members of a species.members of a species.

Golden Rice, A GMO containing Vitamin A

Using Genetics Technology Before Using Genetics Technology Before Recombinant DNA TechnologyRecombinant DNA Technology

Selective Breeding: Choosing Selective Breeding: Choosing organisms that breed together organisms that breed together with the hopes of producing with the hopes of producing “desirable traits” in the next “desirable traits” in the next generation.generation.– Hybrid: mix between two Hybrid: mix between two

distinct things.distinct things.– Inbreeding: mating between Inbreeding: mating between

individuals with similar traits.individuals with similar traits.

It's pretty much my favorite animal. It's like a lion and a tiger mixed... bred for its

skills in magic. -Napoleon Dynamite (in reference to ligers)

Recombinant DNARecombinant DNA

DNA in which genes from 2 DNA in which genes from 2 different sources are linkeddifferent sources are linked– Genetic engineering:Genetic engineering: direct direct

manipulation of genes for manipulation of genes for practical purposespractical purposes

DNA cloning permits production of multiple DNA cloning permits production of multiple copies of a specific gene or other DNA copies of a specific gene or other DNA segmentsegment

To work directly with specific genesTo work directly with specific genes– Scientists have developed methods for Scientists have developed methods for

preparing well-defined, gene-sized pieces of preparing well-defined, gene-sized pieces of DNA in multiple identical copies, a process DNA in multiple identical copies, a process called gene cloningcalled gene cloning

DNA Cloning and Its DNA Cloning and Its Applications: Applications: A PreviewA Preview

Most methods for cloning pieces of Most methods for cloning pieces of DNA in the laboratoryDNA in the laboratory– Share certain general features, such as Share certain general features, such as

the use of bacteria and their the use of bacteria and their plasmidsplasmids

Bacterium

Bacterialchromosome

Plasmid

Cell containing geneof interest

RecombinantDNA (plasmid)

Gene of interest DNA of

chromosome

Recombinatebacterium

Protein harvested

Basic research on protein

Gene of interest

Copies of gene

Basic research on gene

Gene for pestresistance inserted into plants

Gene used to alterbacteria for cleaningup toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growth hormone treatsstunted growth

Protein expressedby gene of interest

3

Gene inserted into plasmid

1

Plasmid put into bacterial cell

2

Host cell grown in culture,to form a clone of cellscontaining the “cloned”gene of interest

3

Basic research and various applications

4

20.1: Using Restriction Enzymes to Make Recombinant DNA

• Bacterial restriction enzymes– Cut DNA molecules at a limited number of

specific DNA sequences, called restriction sites

• Restriction Enzyme Animation

• A restriction enzyme will usually make many cuts in a DNA molecule– Yielding a set of restriction fragments

• The most useful restriction enzymes cut DNA in a staggered way – Producing fragments with “sticky ends” that

can bond with complementary “sticky ends” of other fragments

• DNA ligase is an enzyme– That seals the bonds between restriction

fragments

Restriction site

DNA53 5

3G A A T T CC T T A A G

Sticky end

Fragment from different DNA molecule cut by the same restriction enzyme

One possible combination

Recombinant DNA molecule

G

C T T A AA A T T C

G

A A T T C

C T T A AG

G

G GA A T T C A A T T C

C T T A A G C T T A A G

Using a restriction enzyme and DNA ligase to make recombinant DNA

Restriction enzyme cuts the sugar phosphate backbones at each arrow

1

DNA fragment from another source is added. Base pairing of sticky ends produces various combinations.

2

DNA ligaseseals the strands.

3

Cloning a Eukraryotic Gene in a Bacterial Plasmid

• In gene cloning, the original plasmid is called a cloning vector– Defined as a DNA molecule that can carry

foreign DNA into a cell and replicate there

• Animation

Producing Clones of Cells

1 Isolate plasmid DNA and human DNA.

2 Cut both DNA samples with the same restriction enzyme

3

Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids.

APPLICATION Cloning is used to prepare many copies of a gene of interest for use in sequencing the gene, in producing its encoded protein, in gene therapy, or in basic research.

TECHNIQUE In this example, a human gene is inserted into a plasmid from E. coli. The plasmid contains the ampR gene, which makes E. coli cells resistant to the antibiotic ampicillin. It also contains the lacZ gene, which encodes -galactosidase. This enzyme hydrolyzes a molecular mimic of lactose (X-gal) to form a blue product. Only three plasmids and three human DNA fragments are shown, but millions of copies of the plasmid and a mixture of millions of different human DNA fragments would be present in the samples.

Human DNAfragments

Human cell

Gene of interest

Bacterial cell

ampR gene Bacterial plasmid

Restriction site

Recombinant

DNA plasmids

lacZ gene

RESULTS

Only a cell that took up a plasmid, which has the ampR gene, will reproduce and form a colony. Colonies with nonrecombinant plasmids will be blue, because they can hydrolyze X-gal. Colonies with recombinant plasmids, in which lacZ is disrupted, will be white, because they cannot hydrolyze X-gal. By screening the white colonies with a nucleic acid probe researchers can identify clones of bacterial cells carrying the gene of interest.

Colony carrying non-recombinant plasmid with intact lacZ gene

Bacterialclone

Colony carryingrecombinant plasmidwith disrupted lacZ gene

Recombinantbacteria

4 Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene.

5 Plate the bacteria on agar containingampicillin and X-gal. Incubate untilcolonies grow.

Making HGH using bacteria

Steps for eukaryotic gene cloningSteps for eukaryotic gene cloning(Review)(Review)

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

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

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

Cloning of cells (and foreign Cloning of cells (and foreign genes)genes)

Identification of cell clones Identification of cell clones carrying the gene of interestcarrying the gene of interest

Amplifying DNA Amplifying DNA in Vitroin Vitro: The : The Polymerase Chain Reaction (PCR)Polymerase Chain Reaction (PCR)

The polymerase chain reaction, PCRThe polymerase chain reaction, PCR– Can produce many copies of a specific target Can produce many copies of a specific target

segment of DNAsegment of DNA– Uses primers that bracket the desired Uses primers that bracket the desired

sequencesequence– Uses a heat-resistant DNA polymeraseUses a heat-resistant DNA polymerase

Polymerase chain reaction (PCR)Polymerase chain reaction (PCR)

Copies of any Copies of any piece of DNA piece of DNA without the use of without the use of the body (in vitro)the body (in vitro)

Primers: short Primers: short sequences of sequences of complimentary complimentary DNA to start DNA to start polymerization.polymerization.

Comprehensive Comprehensive website for 20.1website for 20.1

20.2: DNA Analysis20.2: DNA Analysis

Restriction fragment analysis detects Restriction fragment analysis detects DNA differences that affect restriction DNA differences that affect restriction sitessites

Restriction fragment analysisRestriction fragment analysis– Can rapidly provide useful comparative Can rapidly provide useful comparative

information about DNA sequencesinformation about DNA sequences

DNA AnalysisDNA Analysis Gel electrophoresisGel electrophoresis: separates nucleic acids : separates nucleic acids

or proteins on the basis of size or electrical or proteins on the basis of size or electrical charge creating DNA bands of varying lengthcharge creating DNA bands of varying length

20.3 – 20.4: Practical DNA Technology Uses20.3 – 20.4: Practical DNA Technology Uses

Diagnosis of diseaseDiagnosis of disease Human gene therapyHuman gene therapy Pharmaceutical products Pharmaceutical products

(vaccines)(vaccines) ForensicsForensics Animal husbandry Animal husbandry

(transgenic organisms)(transgenic organisms) Genetic engineering in plantsGenetic engineering in plants Ethical concerns?Ethical concerns?

Genetic Engineering in Medicine Genetic Engineering in Medicine and Societyand Society

Very useful for medicine since many disorders are Very useful for medicine since many disorders are caused when the body fails to make proteins.caused when the body fails to make proteins.– Gene therapyGene therapy: putting a healthy copy of a gene : putting a healthy copy of a gene

into the cells of a person with a bad copyinto the cells of a person with a bad copy– Stem Cell ResearchStem Cell Research: use of unspecialized cells : use of unspecialized cells

for replacement of “defective” cellsfor replacement of “defective” cells DNA fingerprintsDNA fingerprints: used to identify an organism: used to identify an organism DNA Fingerprinting ActivityDNA Fingerprinting Activity Using the Evidence ActivityUsing the Evidence Activity

Fig. 20-20

Culturedstem cells

Early human embryoat blastocyst stage

(mammalian equiva-lent of blastula)

Differentcultureconditions

Differenttypes ofdifferentiatedcells

Blood cellsNerve cellsLiver cells

Cells generatingall embryoniccell types

Adult stem cells

Cells generatingsome cell types

Embryonic stem cells

From bone marrowin this example

Genetic Engineering in Genetic Engineering in AgricultureAgriculture

Make plants more Make plants more drought resistant, drought resistant, speed up ripening, speed up ripening, improve nutritional improve nutritional value, and insect value, and insect resistant.resistant.

Transgenic animalsTransgenic animals: : animals with foreign animals with foreign DNA that can do DNA that can do special things.special things.

21.1: Genomics21.1: Genomics

Entire genomes can be mapped at the DNA Entire genomes can be mapped at the DNA levellevel

The Human Genome ProjectThe Human Genome Project– Sequenced the human genomeSequenced the human genome

Scientists have also sequenced genomes of Scientists have also sequenced genomes of other organismsother organisms– Providing important insights of general Providing important insights of general

biological significancebiological significance

DNA SequencingDNA Sequencing

Determination of Determination of nucleotide nucleotide sequencessequences

GenomicsGenomics: the : the study of genomes study of genomes based on DNA based on DNA sequencessequences

Human Genome Human Genome ProjectProject

ActivityActivity