1. Chapter 12
DNA Technology

2. Overview Recombinant DNA technology Application Techniques
DNA profiling and Forensic Science
Genomics and proteomics
Human genome project
Comparing genomes
Genome mapping techniques
Proteomics
Human Gene Therapy
We will learn
Recombinant DNA technology
Application
Techniques
DNA profiling (is used to determine whether two DNA samples come from the same individual) and Forensic Science
Genomics and proteomics
Human genome project
Comparing genomes
Genome mapping techniques
Proteomics
Human Gene Therapy

3. Biology and Society: DNA, Guilt, and Innocence
DNA profiling is the analysis of DNA samples that can be used to determine whether the samples come from the same individual.
Has rapidly revolutionized the field of forensics, the scientific analysis of evidence from crime scenes
DNA profiling can therefore be used in courts to indicate if someone is:
Guilty
Innocent
Figure 12.00
© 2010 Pearson Education, Inc.

4. DNA Technology
Modern laboratory techniques for studying and manipulating genetic material.
Scientists can modify specific genes and move them between organisms like bacteria, plants, and animals.
DNA technology has led to other advances in the:
Creation of genetically modified crops
Identification and treatment of genetic diseases
Biotechnology today means the use of DNA technology, methods for:
- studying and manipulating genetic material,
- modifying specific genes
- moving genes between organisms

5. RECOMBINANT DNA TECHNOLOGY
Biotechnology:
Is the manipulation of organisms or their components to make useful products
Has been used for thousands of years to
Make bread using yeast
Selectively breed livestock for desired traits
Biotechnology today means the use of DNA technology, methods for:
Studying and manipulating genetic material
Modifying specific genes
Moving genes between organisms

6. RECOMBINANT DNA TECHNOLOGY
Recombinant DNA is formed when scientists combine nucleotide sequences (pieces of DNA) from two different sources to form a single DNA molecule.
Recombinant DNA technology is widely used in genetic engineering, the direct manipulation of genes for practical purposes.

7. Recombinant DNA Techniques
Bacteria represent the workhorses of modern biotechnology. To manipulate genes in the laboratory, biologists use bacterial plasmids
small, circular DNA molecules that are separate from the much larger bacterial chromosome
Plasmids:
Can easily incorporate foreign DNA
Are readily taken up by bacterial cells
Can act as vectors, a DNA carriers that move genes from one cell to another
are ideal for gene cloning, the production of multiple identical copies of a gene-carrying piece of DNA
Plasmids
Bacterial
chromosome
Remnant of
bacterium
Colorized TEM
To work with genes in the laboratory, biologists often use bacterial plasmids, small, circular DNA molecules that are separate from the much larger bacterial chromosome.

8. Using recombinant DNA technology to produce useful products
Plasmid
Bacterial cell
Isolate
plasmids.
Some uses
of genes
Gene for pest
resistance
Inserted
into plant
Gene for
toxic-cleanup
bacteria
Genes may be
inserted into
other organisms.
Find the clone with
the gene of interest.
The gene and protein
of interest are isolated
from the bacteria.
Clone the bacteria.
Recombinant bacteria
Bacterial clone
Gene of interest
Recombinant DNA plasmids
Bacteria take up recombinant plasmids.
Harvested
proteins may be
used directly.
of proteins
Protein for
“stone-washing”
jeans
DNA
Cell containing
the gene of interest
Protein for dissolving
clots in heart attack
therapy
DNA.
DNA fragments
from cell
Cut both DNAs
with same
enzyme.
Gene of
interest
Other
genes
Mix the DNAs and
join them together.
Figure 12.8 Using recombinant DNA technology to produce useful products (Step 8)
A rec-DNA is crated by combining a bacterial plasmid and the gene of interest, to understand how these DNA molecules are spliced together, one should know how enzymes cut and paste DNA
How can a researcher obtain DNA that encodes a particular gene of interest?
Recombinant DNA techniques can help biologists produce large quantities of a desired protein

9. Practice
Which of the following is the best definition for recombinant DNA?
A) DNA that results from bacterial conjugation
B) DNA that includes pieces from two different sources
C) an alternate form of DNA that is the product of a mutation
D) DNA that carries a translocation
When plasmids are used to produce a desired protein, the ______.
A) plasmids multiply and produce the protein outside of the bacterium
B) bacterial chromosome is genetically engineered and the plasmid is used to help the bacterium replicate
C) desired gene is inserted into the plasmid and the plasmid is taken up by the bacterium
D) bacterial genome and plasmid are inserted into the genome of the cell containing the desired gene (perhaps the cell of a plant or animal)

10. A Closer Look: Cutting and Pasting DNA with Restriction Enzymes
Recombinant DNA is produced by combining two ingredients a bacterial plasmid and a gene of interest
To combine these ingredients, a piece of DNA must be spliced into a plasmid.
This splicing process can be accomplished by:
using restriction enzymes which cut DNA at specific nucleotide sequences, and
these cuts produce pieces of DNA called restriction fragments with “sticky ends” important for joining DNA from different sources
DNA ligase connects the DNA pieces into continuous strands by forming bonds between adjacent nucleotides.
A restriction enzyme cuts the DNA into fragments between the bases G and A within a recognition sequence, producing a restriction fragment. The staggered cut yield 2-ds DNA fragments with ss ends called sticky ends
A piece of DNA strand from another source is added which has s-s ends identical in base sequence to the sticky ends of green DNA

11. for a restriction enzyme
Cutting and pasting DNA
Recognition sequence
for a restriction enzyme
Restriction
enzyme
Sticky
end
DNA
ligase
Recombinant DNA molecule
A DNA fragment is added
from another source.
A restriction enzyme cuts
the DNA into fragments.
Fragments stick together by
base pairing.
DNA ligase joins the
fragments into strands.
A restriction enzyme cuts the DNA into fragments between the bases G and A within a recognition sequence, producing a restriction fragment. The staggered cut yield 2-ds DNA fragments with ss ends called sticky ends
A piece of DNA strand from another source is added which has s-s ends identical in base sequence to the sticky ends of green DNA

12. A Closer Look: Obtaining the Gene of Interest
How can a researcher obtain DNA that encodes a particular gene of interest?
A “shotgun” approach yields millions of recombinant plasmids carrying many different segments of foreign DNA.
A collection of cloned DNA fragments that includes an organism’s entire genome (a complete set of its genes) is called a genomic library.
Once a genomic library is created, the bacterial clone containing the desired gene is identified using a specific sequence of radioactive nucleotides matching those in the desired gene, called a nucleic acid probe.

13. How a DNA probe tags a gene
Radioactive probe
(single-stranded DNA)
Mix with single-stranded DNA from
various bacterial clones
Single-stranded DNA
Figure How a DNA probe tags a gene
Base pairing indicates the
gene of interest
Figure 12.10

14. Another way to obtain a gene of interest is to:
Cell nucleus
Exon
Intron
Exon
Intron
Exon
DNA of
eukaryotic
gene
Transcription
Another way to obtain a gene of interest is to:
Use reverse transcriptase and
Synthesize the gene by using an mRNA template
RNA
transcript
Introns removed and
exons spliced together
mRNA
Test tube
Isolation of mRNA from
cell and addition of
reverse transcriptase
Reverse
transcriptase
Figure Making a gene from eukaryotic mRNA (Step 5)
Synthesis of cDNA
strand
cDNA strand
being synthesized
Synthesis of second DNA
strand by DNA polymerase
cDNA of gene
without introns
Figure

15. Applications of DNA Technology :
From Humulin to Foods to “Pharm” Animals
By transferring the gene for a desired protein into a bacterium or yeast, proteins that are naturally present in only small amounts can be produced in large quantities.
DNA technology is used to produce medically valuable molecules:
Humulin is human insulin produced by genetically modified bacteria
Was the first recombinant DNA drug approved by the FDA, 1982
Is used today by more than 4 million people with diabetes
Human growth hormone (HGH)
The hormone EPO (Erythropoietin)
which stimulates production of red blood cells
Vaccines,  harmless variant of a disease - causing
microbe (bacteria or virus) that is used to
prevent an infectious disease.

16. Applications of DNA Technology: Genetically Modified Foods
Today, DNA technology is improving productivity of agriculture and quickly replacing traditional plant-breeding programs
Scientists have produced many types of genetically modified (GM) organisms, organisms that have acquired one or more genes by artificial means
If the new gene is from another organism (another species) the recombinant organism is  called a
transgenic organisms.
In the United States today, roughly
one-half of the corn crop and over
three-quarters of the soybean and cotton
crops are genetically modified

17. Practice
Which of the following best defines the term transgenic organism?
A) an organism that is the first of its kind to bear a particular allele
B) an organism in which a genetic defect has been corrected using recombinant DNA therapy
C) an organism containing a gene from another species
D) an organism containing genes from three or more species
Which of these can act as a vector to introduce new genes into a cell?
A) humulin
B) GM
C) PCR
D) plasmids

18. Applications of DNA Technology: GM Foods II
“Golden rice” has been genetically modified to contain beta-carotene used in our bodies to make vitamin A.
Genetically modified rice
Figure 12.5 Genetically modified rice

19. Applications of DNA Technology: “Pharm” Animals
While transgenic plants are used today as commercial products, transgenic whole animals are currently only in the testing phase.
These transgenic sheep carry a gene for a human blood protein
This protein may help in the treatment of cystic fibrosis.
While transgenic animals are currently used to produce potentially useful proteins, none are yet found in our food supply.
It is possible that DNA technology will eventually replace traditional animal breeding.
Transgenic animals raised for the purposes of producing pharmaceuticals are called pharm animals

20. DNA Profiling and Forensic Science
Forensics the scientific analysis of evidence from crime scenes, has been revolutionized by DNA technology.
DNA profiling is used to determine whether two DNA samples come from the same individual.
To produce a DNA profile, scientists compare genetic markers, sequences in the genome that vary from person to person
Investigating Murder, Paternity, and Ancient DNA
DNA profiling (DNA fingerprinting) can be used to establish innocence or guilt of a criminal suspect, identity victims, determine paternity, identify illegally exported animals products, trace the evolutionary history of organisms and contribute to research.
Trace the evolutionary history of organisms: DNA extracted from 27,000 years old Siberian mammoth was 98.6% identical to modern African elephants
A 9,000 years-old cheddar man skeleton provided evidence he was a direct ancestor of a present-day school teacher who lived only half a mile from the cave

21. Overview of DNA profiling
Collect cells
Extract DNA
Cut the DNA in fragments using the same restriction enzyme
4. Separate the fragments using gel electrophoresis
Crime scene
Suspect 1 1
Suspect 2
DNA isolated 2
DNA amplified 3
DNA compared
Figure
Figure Overview of DNA profiling (step 3)

22. Investigating Murder, Paternity, and Ancient DNA
DNA profiling can be used to:
Test the guilt of suspected criminals
Identify tissue samples of victims
Resolve paternity cases
Identify contraband animal products
Trace the evolutionary history of organisms

23. Profiling Techniques
The Polymerase Chain Reaction (PCR)-specific segment of DNA is targeted and copied. Scientists can obtain enough DNA in blood or tissues to allow profiling.
Short Tandem Repeat (STR) Analysis- used to compare genomes in two samples to prove they came from the same person. This is used by a series of short sequences (Repetitive DNA) that is repeated many times one after another.
Gel Electrophoresis - Comparing length of DNA fragments by sorting  macromolecules. 

24. Profiling Techniques: The Polymerase Chain Reaction (PCR)
Is a technique to copy quickly and precisely any segment of DNA and
Can generate enough DNA, from even minute amounts of blood or other tissue, to allow DNA profiling
a single DNA molecule can be replicated in a test tube to make 30 million identical copies in a few hours

25. Polymerase Chain Reaction: DNA Replication in a Test Tube
Exponential Increase in the Number of DNA Molecules each Cycle

26. Profiling Techniques: Short Tandem Repeat (STR) Analysis
How do you test if two samples of DNA come from the same person?
Repetitive DNA:
Makes up much of the DNA that lies between genes in humans and
Consists of nucleotide sequences that are present in multiple copies in the genome
Short tandem repeats (STRs) are
short sequences of DNA that
are repeated many times, tandemly (one after another), in the genome
STR analysis is a method of DNA profiling that
compare the lengths of STR sequences at certain sites in the genome

27. Short tandem repeat (STR) sites
Crime scene DNA
Suspect’s DNA
Same number of
short tandem repeats
Different numbers of
STR site 1
STR site 2
AGAT
GATA
STR sites contain tandem repeats of 4-nucleotide sequences. The number of repetitions at each site vary from individual to individual. Here, both the samples have same number of repeats (7) at first site but different numbers in the second site
STR sites contain tandem repeats of 4-nucleotide sequences. The number of repetitions at each site vary from individual to individual. Here, both the samples have same number of repeats (7) at first site but different numbers in the second site

28. Gel Electrophoresis STR analysis compares the lengths of DNA fragments
Uses gel electrophoresis, a method for sorting macromolecules (usually proteins or nucleic acids) primarily by their electrical charge and Size
Mixture of DNA
fragments of
different sizes
Power
source
Gel
Completed gel
Band of
longest
(slowest)
fragments
shortest
(fastest)

29. Visualizing STR fragment patterns
The DNA fragments are visualized as “bands” on the gel
Difference in the locations of the band reflect the different length of DNA fragments
Provide evidence that crime scene DNA did not come from the suspect
Amplified
crime scene
DNA
suspect’s
Longer
fragments
Shorter
Figure Visualizing STR fragment patterns

30. Practice
Gel electrophoresis separates DNA fragments on the basis of differences in their ______.
A) A:C ratio
B) length
C) G:T ratio
D) pH
STR analysis is a DNA profiling technique that makes use of the fact that different people have
A) different numbers of repeats of short DNA sequences at certain sites in the genome.
B) different alleles for many genes in the genome.
C) different restriction fragments
D) different CODIS DNA sequences

31. Genomics and Proteomics
Genomics  the study of complete sets of genes (genome).
Bacteria were the first targets of genomics.
As of 2009, the genomes of nearly 1000 species have been published, including Baker’s yeast, Mice, Fruit flies, Rice and Sorghum
Genome - Mapping Techniques
The whole- genome shotgun method involves sequencing DNA fragments from an entire genome and then assembling the sequences.
Proteomics
The systematic study of the full set of proteins found in organisms.

32. Table 12.1 Some Important Sequenced Genomes

33. The Human Genome Project
In 1990 an international consortium of government-funded the Human Genome Project.
The goal was to determine the nucleotide sequence of all the DNA in the human genome and identify the location and sequence of every gene
Completed in 2004:
Over 99% of the genome had been determined to % accuracy
3.2 billion nucleotide pairs were identified, about 21,000 genes were found and about 98% of the human DNA was identified as noncoding
The Human Genome Project can help map the genes for specific diseases such as:
Alzheimer’s disease
Parkinson’s disease

34. Genome-Mapping Techniques
Genomes are most often sequenced using whole-genome shotgun method in which:
The entire genome is chopped into fragments using restriction enzymes
The fragments are cloned and sequenced
Computers running specialized mapping software reassemble the millions of overlapping short sequences into a single continuous sequence for every chromosome—an entire genome
Begun in 2006, the Human Variome Project:
Seeks to collect information on all of the genetic variations that affect human health
Chromosome
Chop up with
restriction enzyme
Sequence
fragments
DNA fragments
Align
Reassemble
full sequence

35. HUMAN GENE THERAPY Human gene therapy: Is a recombinant DNA procedure
Normal human
gene isolated
and cloned
gene inserted
into virus
Virus injected
into patient with
abnormal gene
Healthy person
Harmless
virus (vector)
Virus containing
normal human gene
Bone
marrow
Bone of person
with disease
Human gene therapy:
Is a recombinant DNA procedure
Seeks to treat disease by altering the genes of the afflicted person
Often replaces or supplements the mutant version of a gene with a properly functioning one
SCID is a fatal inherited disease caused by a single defective gene that prevents the development of the immune system.
SCID patients quickly die unless treated by bone marrow transplant or Gene therapy.
Since 2000, gene therapy has Cured 22 children with inborn SCID but Unfortunately, caused 4 of the patients to develop leukemia, killing one of these children

36. Practice
The study of the full protein sets that genomes encode is _____.
A) proteomics
B) genomics
C) gene therapy
D) gene cloning
In human gene therapy,
A) normal versions of genes are transferred to patients who carry a mutated allele.
B) harmless bacteria make important proteins for humans that cannot produce these proteins on their own.
C) bacterial plasmids are used to transfer genes to human patients.
D) genetically engineered alleles, usually from other species, replace mutated alleles.

37. Ethics of DNA Technology
Controversy over Genetically Modified Food
The debate about genetically modified crops centers on whether they might harm humans or damage the environment by transferring genes through cross-pollination with other species.
Ethical Questions raised by DNA Technology
Some ethical questions are how far should we take technology?
Some controversies are whether or not it is morally right to know your DNA when you are born and have a DNA profile, and how private that would be.
Also whether or not parents should be able to give their child who has dwarfism a growth hormone to make them grow.
Scientist are still weighing out the positive and negative effects of DNA technology.

38. The Process of Science: Can Genomics Cure Cancer?
Observation: A few patients responded quite dramatically to a new drug, gefitinib, which:
Targets a protein called EGFR found on the surface of cells that line the lungs
Is used to treat lung cancer
Question: Are genetic differences among lung cancer patients responsible for the differences in gefitinib’s effectiveness?
Hypothesis: Mutations in the EGFR gene were causing the different responses to gefitinib.
Prediction: DNA profiling that focuses on the EGFR gene would reveal different DNA sequences in the tumors of responsive patients compared with the tumors of unresponsive patients.
© 2010 Pearson Education, Inc.

39. The Process of Science: Can Genomics Cure Cancer?
Experiment: The EGFR gene was sequenced in the cells extracted from the tumors of:
- Five patients who responded to the drug
- Four who did not
Results: The results were quite striking.
- All five tumors from gefitinib-responsive patients had mutations in EGFR.
- None of the other four tumors did.
The EGFR protein: Fighting cancer with genomics

40. Practice The diagram here summarizes _____. A) how a gene is cloned
B) how a human could be cloned
C) human gene therapy
D) how a vaccine is made
E) how viruses can cause disease
Answer: C
Skill: Knowledge/Comprehension

41. Practice
Please read the following scenario then answer the following question(s).
Molecular biologists have perfected DNA fingerprinting so that it is possible to use the technique to provide evidence to solve crimes and even identify a child's parents. Recently, a U.S. immigrant asked the U.S. Citizenship and Immigration Services for permission to have her young daughter who was living with grandparents in their homeland join her. Her request was denied because there was an apparent mix-up with the child's birth certificate and it could not be used as proof of maternity. Proof is required in cases such as this. The mother requested DNA fingerprinting to make her case. Samples of DNA were taken from the mother (Mom) and daughter (D1) as well as from another daughter (D2) and a son (S) living in the United States with her. Tandem repeat analysis was run on the four samples, and the results are shown here.

42. 1) The results indicate that ______.
A) she is not the mother of the son
B) she is the mother of daughter D1
C) she is not the mother of daughter D1
D) daughter D1 and daughter D2 are identical twins
E) the mother could not be the mother of both daughter D1 and daughter D2