• Sequencing of the human genome was largely completed by 2003
• DNA sequencing has depended on advances in technology, starting with making recombinant
DNA
• In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule
• Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes

• DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products

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

• 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

LE 20-2
Bacterium
Gene inserted into plasmid
Cell containing gene of interest
Bacterial chromosome
Plasmid
Recombinant
DNA (plasmid)
Gene of interest
Plasmid put into bacterial cell
DNA of chromosome
Gene of interest
Copies of gene
Basic research on gene
Recombinant bacterium
Basic research and various applications
Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest
Protein expressed by gene of interest
Protein harvested
Basic research on protein
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
Human growth hormone treats stunted growth

• Bacterial restriction enzymes cut DNA molecules at DNA sequences called restriction sites
• A restriction enzyme usually makes many cuts, yielding restriction fragments
• 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

LE 20-3
Restriction site
DNA 5
3


Restriction enzyme cuts the sugar-phosphate backbones at each arrow.
3

5

Sticky end
DNA fragment from another source is added. Base pairing of sticky ends produces various combinations.
Fragment from different
DNA molecule cut by the same restriction enzyme
DNA ligase seals the strands.
One possible combination
Recombinant DNA molecule

Animation: Restriction Enzymes

• 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

• 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 DNA and human DNA.
Bacterial cell
Cut both DNA samples with the same restriction enzyme.
amp R gene
(ampicillin resistance)
Bacterial plasmid
lacZ gene
(lactose breakdown)
Human cell
Restriction site
Gene of interest
Sticky ends
Human DNA fragments
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids and many nonrecombinant plasmids.
Recombinant DNA plasmids

LE 20-4_2
Isolate plasmid DNA and human DNA.
Bacterial cell
Cut both DNA samples with the same restriction enzyme.
amp R gene
(ampicillin resistance)
Bacterial plasmid
lacZ gene
(lactose breakdown)
Human cell
Restriction site
Gene of interest
Sticky ends
Human DNA fragments
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids and many nonrecombinant plasmids.
Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene.
Recombinant DNA plasmids
Recombinant bacteria

LE 20-4_3
Isolate plasmid DNA and human DNA.
Cut both DNA samples with the same restriction enzyme.
Bacterial cell amp R gene
(ampicillin resistance)
Bacterial plasmid
lacZ gene
(lactose breakdown)
Human cell
Restriction site
Gene of interest
Sticky ends
Human DNA fragments
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids and many nonrecombinant plasmids.
Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene.
Plate the bacteria on agar containing ampicillin and X-gal.
Incubate until colonies grow.
Recombinant DNA plasmids
Recombinant bacteria
Colony carrying nonrecombinant plasmid with intact lacZ gene
Colony carrying recombinant plasmid with disrupted lacZ gene
Bacterial clone

• 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
Colonies containing gene of interest
Master plate
Filter
Solution containing probe
Probe
DNA
Radioactive single-stranded
DNA
Gene of interest
Single-stranded
DNA from cell
Film
A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter.
Filter lifted and flipped over
Hybridization on 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.

• 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

• 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
Cycle 1 yields
2 molecules
Genomic DNA
Denaturation:
Heat briefly to separate DNA strands
5

5

3

3

5

Target sequence
3

3

5

Annealing:
Cool to allow primers to form hydrogen bonds with ends of target sequence
Primers
Extension:
DNA polymerase adds nucleotides to the 3
 end of each primer
New nucleotides
Cycle 2 yields
4 molecules
Cycle 3 yields 8 molecules;
2 molecules
(in white boxes) match target sequence

• Restriction fragment analysis detects differences in the nucleotide sequences of
DNA molecules
• Such analysis can rapidly provide comparative information about DNA sequences

• 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 20-8
Cathode
Power source
Anode
Mixture of DNA molecules of different sizes
Gel
Glass plates
Longer molecules
Shorter molecules

• 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 20-9
Normal b
-globin allele
Ddel
175 bp
Ddel
201 bp
Ddel
Large fragment
Ddel
Sickle-cell mutant b
-globin allele
376 bp Large fragment
Ddel Ddel Ddel
Ddel restriction sites in normal and sickle-cell alleles of b
-globin gene
Normal allele
Sickle-cell allele
Large fragment
376 bp
201 bp
175 bp
Electrophoresis of restriction fragments from normal and sickle-cell alleles

• 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 20-10
DNA + restriction enzyme
Restriction fragments
I II III
I
Normal b
-globin allele
II
Sickle-cell allele
III
Heterozygote
Preparation of restriction fragments.
Gel electrophoresis.
Nitrocellulose paper (blot)
Gel
Sponge
Alkaline solution
Blotting.
Heavy weight
Paper towels
Radioactively labeled probe for b
-globin gene is added to solution in a plastic bag
I II
Paper blot
Hybridization with radioactive probe.
III
Probe hydrogenbonds to fragments containing normal or mutant b
-globin
Fragment from sickle-cell b
-globin allele
Fragment from normal b
-globin allele
I II
Autoradiography.
III
Film over paper blot

• 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

• 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
• Go to video

• 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
Genetic (linkage) mapping
Genes located by FISH
Chromosome bands
Genetic markers
Physical mapping
Overlapping fragments
DNA sequencing

• 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

• Relatively short DNA fragments can be sequenced by the dideoxy chaintermination method
• Inclusion of special dideoxyribonucleotides in the reaction mix ensures that fragments of various lengths will be synthesized

LE 20-12
DNA
(template strand)
5

Primer
3

Deoxyribonucleotides Dideoxyribonucleotides
(fluorescently tagged)
5

DNA polymerase
5

3

DNA (template strand)
Labeled strands
3

3

Direction of movement of 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 fragments short enough for sequencing
Clone the fragments in plasmid or phage vectors
Sequence each fragment
Order the sequences into one overall sequence with computer software

• 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

• 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

• One way to determine function is to disable the gene and observe the consequences
• Using in vitro mutagenesis, mutations are introduced into a cloned gene, altering or destroying its function
• When the mutated gene is returned to the cell, the 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

• 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
Isolate mRNA.
Tissue sample
Make cDNA by reverse transcription, using fluorescently labeled nucleotides.
Apply the cDNA mixture to a microarray, a microscope slide on which copies of singlestranded 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.
mRNA molecules
Labeled cDNA molecules
(single strands)
DNA microarray
Size of an actual
DNA microarray with all the genes of yeast (6,400 spots)

• 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

• 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

• Many fields benefit from DNA technology and genetic engineering

• One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic 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
Restriction sites
RFLP marker
Disease-causing allele
Normal allele

• 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
Retrovirus capsid
Bone marrow cell from patient
Inject engineered cells into patient.
Insert RNA version of normal allele into retrovirus.
Viral RNA
Let retrovirus infect bone marrow cells that have been removed from the patient and cultured.
Viral DNA carrying the normal allele inserts into chromosome.
Bone marrow

• Some pharmaceutical applications of DNA technology:
– Large-scale production of human hormones and other proteins with therapeutic uses
– Production of safer vaccines

• 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 20-17
Defendant’s blood (D)
Blood from defendant’s clothes
Victim’s blood (V)

• 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

• DNA technology is being used to improve agricultural productivity and food quality

• 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

• 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 20-19
Agrobacterium tumefaciens
Ti plasmid
Site where restriction enzyme cuts
DNA with the gene of interest
Recombinant
Ti plasmid
T DNA
Plant with new trait

• 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