Chapter 20

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

PowerPoint Lectures for Biology, Seventh Edition

Neil Campbell and Jane Reece

Lectures by Chris Romero

Chapter 20Chapter 20

DNA Technology and Genomics


Overview: Understanding and Manipulating Genomes

• Sequencing of the human genome was largely completed by 2003

• Recombinant DNA – where DNA sequences from two different sources (species), are combined in vitro into the same DNA molecule


Concept 20.1: DNA cloning permits production of multiple copies of a specific gene or other DNA segment

• GENE CLONING – process of preparing gene-sized pieces of DNA in identical copies

– Allows scientists to work directly with specific genes

LE 20-2LE 20-2Bacterium

Bacterialchromosome

Plasmid

Gene inserted intoplasmid

Cell containing geneof interest

Gene ofinterest DNA of

chromosome

RecombinantDNA (plasmid)

Plasmid put intobacterial cell

Recombinantbacterium

Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest

Protein expressedby gene of interest

Protein harvested

Gene ofinterest

Copies of gene

Basicresearchon gene

Basicresearchon protein

Basic research andvarious applications

Gene for pestresistance insertedinto plants

Gene used to alterbacteria for cleaningup toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growth hor-mone treats stuntedgrowth


Using Restriction Enzymes to Make Recombinant DNA

• Restriction enzyme makes many cuts, yielding restriction fragments

• Fragments with “sticky ends” bond with complementary “sticky ends” of other fragments

• DNA ligase seals the bonds between restriction fragments

LE 20-3LE 20-3Restriction site

DNA 53

35

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

• CLONING VECTOR – the original plasmid that can carry foreign DNA into a cell and it can then be replicated


Producing Clones of Cells

• Cloning a human gene in a bacterial plasmid can be divided into six steps:

1. Vector and gene-source DNA are isolated

2. DNA is inserted into the vector

3. Human DNA fragments are mixed with cut plasmids, and base-pairing takes place

4. Recombinant plasmids are mixed with bacteria

5. The bacteria are plated and incubated

6. Cell clones with the right gene are identified

Animation: Cloning a Gene

LE 20-4_1LE 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_2LE 20-4_2





Humancell

Restrictionsite



interest

Stickyends

Human DNAfragments


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

Recombinantbacteria

LE 20-4_3LE 20-4_3





Humancell

Restrictionsite



interest

Stickyends

Human DNAfragments


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

• A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene

• This process is called nucleic acid hybridization

• An essential step in this process is denaturation of the cells’ DNA, separation of its two strands

LE 20-5LE 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.


• One method of introducing recombinant DNA into eukaryotic cells is electroporation, applying a brief electrical pulse to create temporary holes in plasma membranes

• Alternatively, scientists can inject DNA into cells using microscopic needles

• Once inside the cell, the DNA is incorporated into the cell’s DNA by natural genetic recombination


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

• The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA

• A three-step cycle

– heating (DENATURATION)

– Cooling (ANNEALING)

– replication (EXTENSION)

• Results in an exponentially growing population of identical DNA molecules

LE 20-7LE 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 the nucleotide sequences of DNA molecules

• Such analysis can rapidly provide comparative information about DNA sequences


Gel Electrophoresis and Southern Blotting

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

• This technique uses a gel as a molecular sieve to separate nuclei acids or proteins by size

Video: Biotechnology Lab

LE 20-8LE 20-8

Cathode

Powersource

Anode

Mixtureof DNAmoleculesof differ-ent sizes

Gel

Glassplates

Longermolecules

Shortermolecules


• A technique called Southern blotting combines gel electrophoresis with nucleic acid hybridization

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

LE 20-10LE 20-10

DNA + restriction enzyme Restrictionfragments

Normal-globinallele

Sickle-cellallele

Heterozygote

Preparation of restriction fragments. Gel electrophoresis. Blotting.

Nitrocellulosepaper (blot)

Gel

Sponge

Alkalinesolution

Papertowels

Heavyweight

Hybridization with radioactive probe.

Radioactivelylabeled 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.

Film overpaper blot


Restriction Fragment Length Differences as Genetic Markers

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

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

• RFLPs are detected by Southern blotting


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

• The most ambitious mapping project to date has been the sequencing of the human genome

• Officially begun as the Human Genome Project in 1990, the 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

• The first stage in mapping a large genome is constructing a linkage map of several thousand genetic markers throughout each chromosome

• The order of markers and relative distances between them are based on recombination frequencies

LE 20-11LE 20-11

Cytogenetic map

Genes locatedby FISH

Chromosomebands

Geneticmarkers

Genetic (linkage)mapping

Physical mapping

Overlappingfragments

DNA sequencing


Physical Mapping: Ordering DNA Fragments

• A physical map is constructed by cutting a DNA molecule into many short fragments and arranging them in order by identifying overlaps

• Physical mapping gives the actual distance in base pairs between markers


DNA Sequencing

• Relatively short DNA fragments can be sequenced by the dideoxy chain-termination method

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

LE 20-12LE 20-12DNA(template strand)

5

3

Primer3

5

DNApolymerase

Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)

3

5DNA (templatestrand)

Labeled strands3

Directionof movementof strands

Laser Detector


• Linkage mapping, physical mapping, and DNA sequencing represent the overarching strategy of the 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 20-13LE 20-13

Cut 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


Determining Gene Function

• Disable the gene and observe the consequences

• In vitro mutagenesis mutations are introduced into a cloned gene, altering or destroying its function

– Mutated gene is returned to the cell to determine gene’s function by organisms phenotype


Studying Expression of Interacting Groups of Genes

• Automation : Use DNA microarray assays to test many gene locations at once

– compare patterns of gene expression in different tissues, at different times, or under different conditions

LE 20-14LE 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

• Provide information in many fields of biology

• Can determine organisms evolutionary history

• Correlate research with lower organisms to human biology


Future Directions in Genomics

• Genomics is the study of entire genomes

• Proteomics is the 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

– Genetic disorders

– Human Gene Therapy

– Pharmaceuticals

– Forensic Science

– Environmental Cleanup

– Agriculture – transgenic organisms – animals and plants


SUMMARY

• Describe recombinant DNA and how a gene is cloned in bacteria – plasmids, restriction enzymes, vector, nucleic acid hybridization, clone isolation

• Describe amplification of DNA segments in vitro using PCR

• Describe gel electrophoresis, Southern blotting, and RFLPs.

• Describe methods in genomic mapping – cytogenic map, linkage map, physical mapping, DNA sequencing, dideoxy chain-termination method, random DNA fragments

• Determining protein and gene function;

• Practical applications of all of this technology

Chapter 20

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