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

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

DNA Technology


• Sequencing of the human genome was largely completed by 2003

• In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule


• An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes


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

• To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called gene cloning


DNA Cloning and Its Applications: A Preview

• Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids

• Cloned genes are useful for making copies of a particular gene and producing a gene product


Using Restriction Enzymes to Make Recombinant DNA• Bacterial restriction enzymes cut DNA molecules at DNA

sequences called restriction sites

• A restriction enzyme usually makes many cuts, yielding restriction fragments

• Were discovered in bacteria to protect them from viruses dna takeover.

• Methylation of cut site on bacterial DNA protects bacteria from getting cut up to

• The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary “sticky ends” of other fragments

• DNA ligase is an enzyme that seals the bonds between restriction fragments


Restriction enzymes

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


Animation: Restriction Enzymes


How do you get DNA sequences of choice?

• Bacterial DNA has not introns in its genome.

• Need to create DNA without introns.

• How do you do that.

• Take mRNA and use reverse transcriptase to create DNA without introns.

• Replicate that DNA to be inserted in Bacteria.


Cloning a Eukaryotic Gene in a Bacterial Plasmid

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

• A cloning vector is a DNA molecule that can carry foreign DNA into a cell and replicate there

LE 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


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_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





Humancell

Restrictionsite



interest

Stickyends

Human DNAfragments


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

Recombinantbacteria

LE 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 20-5

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.

LE 20-6

Bacterialclones

Recombinantplasmids

Recombinantphage DNA

or

Foreign genomecut up withrestrictionenzyme

Phageclones

Plasmid library Phage library


Bacterial Expression Systems

• Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells

• To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active prokaryotic promoter


• 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, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

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


• http://www.dnalc.org/ddnalc/resources/pcr.html

• http://spine.rutgers.edu/cellbio/flash/pcr.htm

• http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf

http://www.dnalc.org/ddnalc/resources/pcr.html

http://spine.rutgers.edu/cellbio/flash/pcr.htm

http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf

http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf


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


ELECTROPHORESIS

• http://www.dnalc.org/ddnalc/resources/electrophoresis.html

• http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html

• http://www.ndsu.nodak.edu/instruct/mcclean/plsc431/linkage/linkage6.htm

http://www.dnalc.org/ddnalc/resources/electrophoresis.html

http://www.dnalc.org/ddnalc/resources/electrophoresis.html

http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html

http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html

http://www.ndsu.nodak.edu/instruct/mcclean/plsc431/linkage/linkage6.htm

http://www.ndsu.nodak.edu/instruct/mcclean/plsc431/linkage/linkage6.htm

LE 20-8

Cathode

Powersource

Anode

Mixtureof DNAmoleculesof differ-ent sizes

Gel

Glassplates

Longermolecules

Shortermolecules


• In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis

• Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene

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

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


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 blottingGenetic (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 20-11

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


Sanger method of sequencing

http://www.rvc.ac.uk/review/DNA_1/2_DNA_Sequencing.cfm

http://smcg.ccg.unam.mx/enp-unam/03-EstructuraDelGenoma/animaciones/secuencia.swf

http://www.dnalc.org/ddnalc/resources/sangerseq.html

http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter15/animation_quiz_1.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 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 20-13

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


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


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)


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


Medical Applications

• One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases


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

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

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Transcript of Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings DNA Technology.