Chapter 20

Chapter 20DNA Technology & Genomics

• Genetic Engineering– manipulation of DNA– if you are going to engineer DNA & genes &

organisms, then you need a set of tools to work with

– this unit is a survey of those tools…

Biotechnology Today

Our tool kit…

Understanding and Manipulating Genomes

• DNA sequencing has depended on advances in technology, starting with making recombinant DNA

• Recombinant DNA - nucleotide sequences from 2 different sources are combined in vitro into same DNA molecule

• Methods for making recombinant DNA are central to genetic engineering, direct manipulation of genes for practical purposes

20.1: DNA cloning permits production of multiple copies of a specific gene

To work with specific genes, scientists prepare gene-sized pieces of DNA in identical copies - gene cloning

Using Restriction Enzymes to Make Recombinant DNA

• Bacterial restriction enzymes - cut DNA molecules at DNA sequences called restriction sites

• Restriction enzyme usually makes many cuts, yielding restriction fragments

• Most useful restriction enzymes cut DNA in a staggered way - producing fragments with “sticky ends” - bond with complementary “sticky ends” of other fragments

• DNA ligase - enzyme that seals bonds between restriction fragments

LE 20-3Restriction site

DNA 5¢3¢

3¢5¢

Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow.

One possible combination

DNA fragment from anothersource is added. Base pairingof sticky ends producesvarious combinations.

Fragment from differentDNA molecule cut by thesame restriction enzyme

DNA ligaseseals the strands.

Recombinant DNA molecule

Sticky end

Cloning a Eukaryotic Gene in a Bacterial Plasmid

• In gene cloning, original plasmid is called a cloning vector

• Cloning vector - DNA molecule that can carry foreign DNA into a cell and replicate there

Producing Clones of Cells• Cloning human gene in bacterial plasmid:

1. Vector and gene-source DNA are isolated2. DNA is inserted into vector3. Human DNA fragments are mixed with cut plasmids,

and base-pairing takes place4. Recombinant plasmids are mixed with bacteria5. The bacteria are plated and incubated6. Cell clones with the right gene are identified

LE 20-4_1

Isolate plasmid DNAand human DNA.

Cut both DNA samples withthe same restriction enzyme.

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

Bacterial cell lacZ gene(lactosebreakdown)

Humancell

Restrictionsite

ampR gene(ampicillinresistance)

Bacterialplasmid Gene of

interest

Stickyends

Human DNAfragments

Recombinant DNA plasmids

LE 20-4_2

Isolate plasmid DNAand human DNA.

Cut both DNA samples withthe same restriction enzyme.

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

Bacterial cell lacZ gene(lactosebreakdown)

Humancell

Restrictionsite

ampR gene(ampicillinresistance)

Bacterialplasmid Gene of

interest

Stickyends

Human DNAfragments

Recombinant DNA plasmids

Introduce the DNA into bacterial cellsthat have a mutation in their own lacZgene.

Recombinantbacteria

LE 20-4_3

Isolate plasmid DNAand human DNA.

Cut both DNA samples withthe same restriction enzyme.

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

Bacterial cell lacZ gene(lactosebreakdown)

Humancell

Restrictionsite

ampR gene(ampicillinresistance)

Bacterialplasmid Gene of

interest

Stickyends

Human DNAfragments

Recombinant DNA plasmids

Introduce the DNA into bacterial cellsthat have a mutation in their own lacZgene.

Recombinantbacteria

Plate the bacteria on agarcontaining ampicillin and X-gal.Incubate until colonies grow.

Colony carrying non-recombinant plasmidwith intact lacZ gene

Colony carryingrecombinantplasmid withdisrupted lacZ gene

Bacterialclone

Identifying Clones Carrying a Gene of Interest

• Clone carrying gene of interest can be identified with a nucleic acid probe

• Called nucleic acid hybridization

• Radioactive or fluorescent probes are engineered to be complimentary to a target sequence

• First, denature of cells’ DNA

LE 20-5

Master plate

Filter

Solutioncontainingprobe

Filter liftedand flipped over

Radioactivesingle-strandedDNA

ProbeDNA

Gene ofinterest

Single-strandedDNA from cell

Film

Hybridizationon filter

Master plate

Coloniescontaininggene ofinterest

A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter.

The filter is treated to break open the cells and denature their DNA; the resulting single-stranded DNA molecules are treated so that they stick to the filter.

The filter is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography).

After the developed film is flipped over, the reference marks on the film and master plate are aligned to locate colonies carrying the gene of interest.

Storing Cloned Genes in DNA Libraries• Genomic library - collection of recombinant

vector clones produced by cloning DNA fragments from an entire genome

• Complementary DNA (cDNA) library - made by cloning DNA made in vitro by reverse transcription of all mRNA produced by a particular cell

• cDNA library - represents only part of genome—only subset of genes transcribed into mRNA in original cells

• Solves problem of prokaryotes not having machinery to remove introns

Bacterial Expression Systems• Several technical difficulties hinder expression

of cloned eukaryotic genes in bacterial host cells

• Have to overcome differences in promoters and other DNA control sequences

Eukaryotic Cloning and Expression Systems• Use of yeast artificial

chromosomes (YACs) as vectors helps avoid gene expression problems

• YACs - behave normally in mitosis and can carry more DNA than a plasmid

• Eukaryotic hosts can provide posttranslational modifications that many proteins require

Introducing recombinant DNA into eukaryotic cells:• electroporation, - applying a brief electrical

pulse to create temporary holes in plasma membranes

• inject DNA into cells using microscopic needles

Polymerase Chain Reaction (PCR)• Polymerase chain reaction (PCR) - produce

many copies of specific target segment of DNA• 3-step cycle: heating, cooling, and replication• chain reaction that produces an exponentially

growing population of identical DNA molecules• http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf

LE 20-7

Genomic DNA

Targetsequence

5¢

3¢

3¢

5¢

5¢

3¢

3¢

5¢

Primers

Denaturation:Heat brieflyto separate DNAstrands

Annealing:Cool to allowprimers to formhydrogen bondswith ends oftarget sequence

Extension:DNA polymeraseadds nucleotides tothe 3¢ end of eachprimer

Cycle 1yields

2molecules

Newnucleo-

tides

Cycle 2yields

4molecules

Cycle 3yields 8

molecules;2 molecules

(in white boxes)match target

sequence

Concept 20.2: Restriction fragment analysis detects DNA differences that affect restriction

sites

• Restriction fragment analysis - detects differences in nucleotide sequences of DNA molecules

• provide comparative information about DNA sequences

Gel Electrophoresis and Southern Blotting

• One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis

• Uses a gel as a molecular sieve to separate nuclei acids or proteins by size

• DNA is negatively charged and moves towards a positive charge when placed in an electrical field

• restriction fragment analysis - fragments of DNA molecule are sorted by gel electrophoresis

• Useful for comparing two different DNA molecules, such as two alleles for a gene

RFLP Analysis

Restriction Fragment Length Differences as Genetic Markers

• Restriction fragment length polymorphisms (RFLPs, or Rif-lips) - differences in DNA sequences on homologous chromosomes that result in restriction fragments of different lengths

• A RFLP can serve as genetic marker for a particular location (locus) in the genome

• RFLPs are detected by Southern blotting

LE 20-9Normal b-globin allele

175 bp 201 bp Large fragment

Sickle-cell mutant b-globin allele

376 bp Large fragment

Ddel Ddel Ddel Ddel

Ddel Ddel Ddel

Ddel restriction sites in normal and sickle-cell alleles of-globin gene

Normalallele

Sickle-cellallele

Largefragment

376 bp201 bp175 bp

Electrophoresis of restriction fragments from normaland sickle-cell alleles

Uses: Evolutionary relationships• Comparing DNA samples from different

organisms to measure evolutionary relationships

–

+

DNA

1 32 4 5 1 2 3 4 5

turtle snake rat squirrel fruitfly

Uses: Medical diagnostic• Comparing normal allele to disease allele

chromosome with disease-causing allele 2

chromosomewith normal allele 1 –

+

allele 1allele 2

DNA

Example: test for Huntington’s disease

Uses: Forensics• Comparing DNA sample from crime scene

with suspects & victim

–

+

S1

DNA

S2 S3 Vsuspects crime

scene sample

DNA fingerprints• Comparing blood

samples on defendant’s clothing to determine if it belongs to victim– DNA fingerprinting– comparing DNA banding

pattern between different individuals

– ~unique patterns

• Southern blotting - combines gel electrophoresis with nucleic acid hybridization

• Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to DNA immobilized on a “blot” of gel

• http://highered.mcgraw-hill.com/olc/dl/120078/bio_g.swf

LE 20-10

DNA + restriction enzyme Restrictionfragments

I Normal-globinallele

I Sickle-cellallele

I Heterozygote

Preparation of restriction fragments. Gel electrophoresis. Blotting.

I I I Nitrocellulosepaper (blot)

Gel

Sponge

Alkalinesolution

Papertowels

Heavyweight

Hybridization with radioactive probe.

I I IRadioactivelylabeled probefor -globingene is addedto solution ina plastic bag

Paper blot

Probe hydrogen-bonds to fragmentscontaining normalor mutant -globin

Fragment fromsickle-cell-globin allele

Fragment fromnormal -globinallele

Autoradiography.

I I I

Film overpaper blot

Concept 20.3: Entire genomes can be mapped at the DNA level

Most ambitious mapping project to date has been the sequencing of the human genome

Officially begun as Human Genome Project in 1990, sequencing was largely completed by 2003

Scientists have also sequenced genomes of other organisms, providing insights of general biological significance

Genetic (Linkage) Mapping: Relative Ordering of Markers

1st stage is constructing linkage map of several thousand genetic markers throughout each chromosome

Order of markers and relative distances between them are based on recombination frequencies

LE 20-11Cytogenetic map

Genes locatedby FISH

Chromosomebands

Geneticmarkers

Genetic (linkage)mapping

Physical mapping

Overlappingfragments

DNA sequencing

Physical Mapping: Ordering DNA Fragments

Physical map - constructed by cutting DNA molecule into many short fragments and arranging them in order by identifying overlaps

Physical mapping gives actual distance in base pairs between markers

DNA Sequencing Relatively short DNA fragments can be

sequenced by dideoxy chain-termination method

Inclusion of special dideoxyribonucleotides in reaction mix ensures that fragments of various lengths will be synthesized

http://www.dnalc.org/resources/animations/cycseq.html

LE 20-12DNA(template strand)

5¢

3¢

Primer3¢

5¢

DNApolymerase

Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)

3¢

5¢DNA (templatestrand)

Labeled strands3¢

Directionof movementof strands

Laser Detector

Linkage mapping, physical mapping, and DNA sequencing represent overarching strategy of Human Genome Project

An alternative approach to sequencing genomes starts with sequencing random DNA fragments

Computer programs then assemble overlapping short sequences into one continuous sequence

LE 20-13Cut the DNA from many copies of an entire chromosome into overlapping frag-ments short enough for sequencing

Clone the fragments in plasmid or phagevectors

Sequence each fragment

Order the sequences into one overall sequence with computer software

Concept 20.4: Genome sequences provide clues to important biological questions

In genomics, scientists study whole sets of genes and their interactions

Genomics is yielding new insights into genome organization, regulation of gene expression, growth and development, and evolution

Identifying Protein-Coding Genes in DNA Sequences

Computer analysis of genome sequences helps identify sequences likely to encode proteins

The human genome contains about 25,000 genes, but the number of human proteins is much larger

Comparison of sequences of “new” genes with those of known genes in other species may help identify new genes

NOVA Science Now: Public Genomes

Determining Gene Function One way to determine function is to disable gene

and observe consequences Using in vitro mutagenesis, mutations are

introduced into cloned gene, altering or destroying its function

When mutated gene is returned to cell, normal gene’s function might be determined by examining the mutant’s phenotype

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

Studying Expression of Interacting Groups of Genes

Automation has allowed scientists to measure expression of thousands of genes at one time using DNA microarray assays

DNA microarray assays - compare patterns of gene expression in different tissues, at different times, or under different conditions

LE 20-14

Make cDNA by reverse transcription, using fluorescently labeled nucleotides.

Apply the cDNA mixture to a microarray, a microscope slide on which copies of single-stranded DNA fragments from the organism’s genes are fixed, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray.

Rinse off excess cDNA; scan microarray for fluorescent. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample.

Isolate mRNA.Tissue sample

mRNA molecules

Labeled cDNA molecules(single strands)

DNAmicroarray

Size of an actualDNA microarraywith all the genesof yeast (6,400 spots)

Comparing Genomes of Different Species Comparative studies of genomes from related

and widely divergent species provide information in many fields of biology

The more similar the nucleotide sequences between two species, the more closely related these species are in their evolutionary history

Comparative genome studies confirm the relevance of research on simpler organisms to understanding human biology

NOVA Science NOW: Autism Video

Future Directions in Genomics Genomics - study of entire genomes Proteomics - systematic study of all proteins

encoded by a genome Single nucleotide polymorphisms (SNPs) - provide

markers for studying human genetic variation

Concept 20.5: The practical applications of DNA technology affect our lives in many ways

Many fields benefit from DNA technology and genetic engineering

Medical Applications One benefit of DNA technology is identification of

human genes in which mutation plays a role in genetic diseases

Gene testing videohttp://www.pbs.org/wgbh/nova/body/public-genomes.html

Diagnosis of Diseases Scientists can diagnose many human genetic

disorders by using PCR and primers corresponding to cloned disease genes, then sequencing the amplified product to look for the disease-causing mutation

Even when a disease gene has not been cloned, presence of an abnormal allele can be diagnosed if a closely linked RFLP marker has been found

LE 20-15

DNA

RFLP marker

Disease-causingallele

Normal allele

Restrictionsites

Human Gene Therapy Gene therapy is the alteration of an afflicted

individual’s genes Gene therapy holds great potential for treating

disorders traceable to a single defective gene Vectors are used for delivery of genes into cells Gene therapy raises ethical questions, such as

whether human germ-line cells should be treated to correct the defect in future generations

LE 20-16

Cloned gene

Retroviruscapsid

Bonemarrowcell frompatient

Inject engineeredcells into patient.

Insert RNA version of normal alleleinto retrovirus.

Viral RNA

Let retrovirus infect bone marrow cellsthat have been removed from thepatient and cultured.

Viral DNA carrying the normalallele inserts into chromosome.

Bonemarrow

Pharmaceutical Products• Some pharmaceutical applications of DNA

technology:– Large-scale production of human hormones and

other proteins with therapeutic uses– Production of safer vaccines

Forensic Evidence DNA “fingerprints” obtained by analysis of tissue

or body fluids can provide evidence in criminal and paternity cases

A DNA fingerprint is a specific pattern of bands of RFLP markers on a gel

The probability that two people who are not identical twins have the same DNA fingerprint is very small

Exact probability depends on the number of markers and their frequency in the population

LE 20-17Defendant’sblood (D)

Blood from defendant’sclothes

Victim’sblood (V)

Environmental Cleanup Genetic engineering can be used to modify the

metabolism of microorganisms Some modified microorganisms can be used to

extract minerals from the environment or degrade potentially toxic waste materials

Agricultural Applications DNA technology is being used to improve

agricultural productivity and food quality

Animal Husbandry and “Pharm” Animals Transgenic organisms are made by introducing

genes from one species into the genome of another organism

Transgenic animals may be created to exploit the attributes of new genes (such as genes for faster growth or larger muscles)

Other transgenic organisms are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use

Genetic Engineering in Plants Agricultural scientists have endowed a number

of crop plants with genes for desirable traits The Ti plasmid is the most commonly used

vector for introducing new genes into plant cells

LE 20-19Agrobacterium tumefaciens

Tiplasmid

Site whererestrictionenzyme cuts

DNA withthe geneof interest

T DNA

RecombinantTi plasmid

Plant withnew trait

Safety and Ethical Questions Raised by DNA Technology

Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures

Most public concern about possible hazards centers on genetically modified (GM) organisms used as food