2. Key Concepts
Enzymes that cut DNA at specific locations and other enzymes that piece DNA segments back together allow biologists to move genes from one place to another.
Biologists can obtain many identical copies of a gene by: (1) inserting it into a bacterial cell that copies the gene each time the cell divides or (2) by conducting a polymerase chain reaction (PCR).

3. Key Concepts
The sequence of bases in a gene can be determined by dideoxy sequencing.
If individuals with a certain phenotype also tend to share a genetic marker (a known site in DNA that is unrelated to the phenotype), the gene responsible for the phenotype is likely to be near that marker.
Researchers are attempting to insert genes into humans to cure genetic diseases. Efforts to insert genes into plants have been much more successful.

4. Introduction
Manipulation of DNA sequences in organisms is known as genetic engineering, and techniques used to engineer genes are called recombinant DNA technology.

5. The Effort to Cure Pituitary Dwarfism
Pituitary dwarfism results from the lack of production of growth hormone, encoded by the GH1 gene.
Pituitary dwarfism type I is an autosomal recessive trait. Affected individuals have two copies of the defective allele.
Humans affected by pituitary dwarfism grow slowly, reaching a maximum adult height of about 4 feet.

7. Why Did Early Efforts to Treat the Disease Fail?
Early trials showed that people with pituitary dwarfism could be treated successfully with growth hormone therapy, but only if the protein came from humans.
Growth hormone purified from the pituitary glands of human cadavers is scarce and expensive.
Human treatment with growth hormone from cadavers has been banned due to possible contamination with prions—protein particles that have been implicated as the cause of various neurodegenerative disorders.

8. Engineering a Safe Supply of Growth Hormone
The recombinant DNA strategy for producing human growth hormone involved cloning the human gene, introducing the gene into bacteria, and having the recombinant cells produce the hormone.

9. Using Reverse Transcriptase to Produce cDNAs
The enzyme reverse transcriptase can synthesize DNA from an RNA template.
Researchers used reverse transcriptase to make complementary DNA (cDNA) from mRNA isolated from pituitary cells. (cDNA is any DNA made from an RNA template.)
They then used DNA cloning—the process of producing many identical copies of a gene—to copy the cDNAs for analysis to determine which encoded the growth hormone protein.

11. Using Plasmids in Cloning
Plasmids are small, circular DNA molecules often found in bacteria. They replicate independently of the chromosome.
Plasmids can serve as a vector—a vehicle for transferring recombinant genes to a new host.
If a recombinant plasmid can be inserted into a bacterial or yeast cell, the foreign DNA will be copied and transmitted to new cells as the host cell grows and divides. In this way plasmids can be used to produce millions of identical copies of specific genes.

12. Cutting and Pasting DNA
Restriction endonucleases are bacterial enzymes that cut DNA at specific base sequences called recognition sites.
The first step in cloning genes into plasmids is to cut the plasmid and the cDNA with the same restriction endonuclease.
Restriction endonucleases often make staggered cuts in the DNA, resulting in sticky ends, complementary single-stranded ends.
The sticky ends of the plasmids and cDNAs will bind by complementary base pairing.
DNA ligase then seals the recombinant pieces of DNA together.

14. The Importance of the Creation of Sticky Ends
If restriction sites in different DNA sequences are cut with the same restriction endonuclease, the presence of the same sticky ends in both samples of DNA allows the resulting fragments to be spliced together by complementary base pairing. This is the essence of recombinant DNA technology—the ability to create novel combinations of DNA sequences by cutting specific sequences and pasting them into new locations.

15. Transformation
If a recombinant plasmid can be inserted into a bacterial or yeast cell, the foreign DNA will be copied and transmitted to new cells as the host cell grows and divides. In this way, researchers can obtain millions or billions of copies of specific genes.
Plasmid vectors can be introduced into bacteria by transformation, the process of taking up DNA from the environment and incorporating it into the genome.

16. Producing a cDNA Library
A DNA library is a collection of transformed bacterial cells, each containing a vector with an inserted gene.
A cDNA library is a collection of bacterial cells, each containing a vector with one cDNA.
A genomic library is made up of cloned DNA fragments representing an entire genome.
DNA libraries are important because they give researchers a way to store information from a particular cell type or genome in an accessible form.

18. Screening a DNA Library
A DNA probe is a single-stranded fragment of a known gene that binds a complementary sequence in the sample of DNA being analyzed.
A DNA probe must be labeled so it can be found after it has bound the target sequence.

20. Screening a DNA Library
The growth hormone researchers inferred the approximate sequence for the GH1 gene from the amino acid sequence of human growth hormone.
They constructed a probe based on this inferred sequence and radioactively labeled it.
They then used this probe to screen a cDNA library for the plasmid containing the GH1 cDNA.

24. Mass-Producing Growth Hormone
Once the researchers found the human growth hormone cDNA, they cloned it in a plasmid under the control of a bacterial promoter.
The transformed E. coli cells produced human growth hormone that could be isolated and purified in large quantities.

25. Ethical Concerns over Recombinant Growth Hormone
The increased supply of growth hormone led to its use to treat children who were short but not did not suffer from pituitary dwarfism.
The U.S. Food and Drug Administration has now approved use of the hormone only for children projected to reach adult heights of less than 5'3" for males and 4'11" for females.

26. The Polymerase Chain Reaction
The polymerase chain reaction (PCR) is an in vitro DNA synthesis reaction in which a specific DNA sequence is replicated over and over again.
This technique generates many identical copies of a particular DNA sequence.

27. Requirements of PCR
PCR is possible only when DNA sequence information surrounding the gene of interest is available, because PCR requires primers that match sequences on either side of the gene.
One primer is complementary to a sequence on one strand upstream of the target DNA and the other primer is complementary to a sequence on the other strand downstream of the target.
The primers will bind to single-stranded target DNA.

29. The Steps of Polymerase Chain Reaction
1. A reaction mix containing dNTPs, a DNA template, copies of the two primers, and Taq polymerase.
2. Denaturation – heating the mixture to 95°C separates the two strands of the DNA.
3. Primer annealing – cooling the mixture allows the primers to bond, or anneal, to complementary sections of single-stranded target DNA.
4. Extension – heating the mixture to 72°C causes the Taq polymerase to synthesize the complementary DNA strand from the dNTPs, starting at the primer.
5. Steps 2–4 are continually repeated to yield the necessary number of copies.

31. PCR in Action: Studying Fossil DNA
Svante Pääbo and colleagues used PCR to compare DNA sequences from a 30,000-year-old Homo neanderthalensis fossil with modern Homo sapiens DNA to analyze how similar the two species are.
These sequences proved to be highly distinct and so support the hypothesis that Neanderthals never interbred with modern humans.
Because the complete genomes of a wide array of organisms have now been sequenced, researchers can find appropriate primer sequences to use in cloning almost any target gene by PCR.

32. Dideoxy DNA Sequencing
Determining a cloned gene’s base sequence is useful for understanding more about the gene’s function.
Fredrick Sanger developed dideoxy sequencing as a method for determining DNA sequence.
Sanger had to link three important insights to make his sequencing strategy work.
The method is based on an in vitro DNA synthesis reaction.
The following site contains a great animation on Sanger’s process:

33. Dideoxy DNA Sequencing
Dideoxy sequencing is carried out by adding both dideoxynucleotide triphosphates (ddNTPs) and deoxyribonucleotide triphosphates (dNTPs) to the synthesis reactions.
ddNTPs are identical to dNTPs except that they lack the 3' hydroxyl group.
Because of this lack, DNA polymerization stops once a ddNTP is added to a growing strand.

34. Dideoxy DNA Sequencing
In the original technique, four separate reactions were performed, each containing all four dNTPs and one of the four ddNTPs.
When the four reactions were separated, side by side, by gel electrophoresis, they revealed the DNA sequence.
The current technique uses fluorescent markers for each ddNTP to simplify the DNA sequencing.
This allows DNA to be sequenced with one dideoxy reaction instead of four.

38. “Next Generation” Sequencing
New approaches to sequencing now make it possible to compare sequences from individuals of a particular species much faster and more cheaply than the dideoxy method.
In pyrosequencing, the pyrophosphate that is released after a DNA polymerase adds a dNTP to a growing DNA strand is detected. These pyrophosphates drive a set of other reactions that result in luminescence, which can be detected.

39. How Was the Huntington's Disease Gene Found?
Huntington’s disease is a rare but devastating neurodegenerative disorder that is eventually fatal.
An analysis of pedigrees from affected families suggested that the trait results from a single, autosomal dominant allele.
This means that sons or daughters of a Huntington's sufferer have a 50 percent chance of receiving the disease allele and developing the illness.
Researchers set out to identify the gene or genes involved and to document that one or more genes are altered in affected individuals.

40. Locating Specific Genes
To locate the gene or genes associated with a particular phenotype, such as a disease, researchers traditionally started with a genetic map (or linkage map or meiotic map).
More recently, biologists have begun using a physical map of the genome. A physical map records the absolute position of a gene—in numbers of base pairs—along a chromosome.

41. Using Genetic Markers
Genetic maps are valuable because they contain genetic markers (genes or other loci that have known locations).
Each genetic marker provides a landmark at a position along a chromosome that is known relative to other markers.
Genetic markers must be polymorphic in order to be useful— in other words, the phenotype associated with the marker must be variable.
DNA samples from affected families can be analyzed with genetic markers to follow the inheritance of specific DNA regions.

42. Using Genetic Markers
If you observe that a certain marker and a certain phenotype are almost always inherited together, it is logical to conclude that the genes involved are physically close to each other on the same chromosome—meaning that they are closely linked.
To locate specific disease genes, researchers must find a large number of affected and unaffected people, and then locate a genetic marker that occurs in the affected individuals but not in the unaffected people.

43. Using Genetic Markers
Single nucleotide polymorphisms (SNPs) are sites in DNA where some individuals in the population have different bases.
SNPs can be used as genetic markers.

45. Pinpointing the Huntington’s Disease Defect
The researchers sequenced exons in the location of the Huntington's disease gene from affected and unaffected individuals to pinpoint specific bases that differ between the two groups.
Individuals with Huntington's disease have an unusual number of CAG codons at the 5' end of a particular gene.
They called this gene IT15 and its protein product huntingtin. Huntingtin is involved in the early development of nerve cells.

46. What Are the Benefits of Finding a Disease Gene?
There are three major benefits of successful disease-gene hunts:
Improved understanding of the phenotype.
Possibilities for new types of therapy.
Additional genetic testing.

47. Improved Understanding of the Phenotype
Once a disease gene is found, the relationship between the gene and the resulting disease phenotype is better understood.
In Huntington's patients, huntingtin protein forms aggregates in the brain, eventually causing neurons to die.
These aggregates are thought to be a direct consequence of changes in the number of CAG repeats in the IT15 gene.

48. Therapy
Therapies can be discovered by using transgenic animals that have had the defective allele introduced into their genome.
If the animals exhibit disease symptoms that parallel those of a human disease they are said to provide an animal model of the disease.
Researchers can use animal models to test possible treatments.

49. Three Types of Genetic Tests for Genetic Diseases
Carrier testing can determine if an individual carries a defective allele.
Prenatal testing can determine if a fetus has a genetic disease by analyzing some of its cells early in gestation.
Adult testing can inform individuals if they are more likely to develop certain diseases, such as breast cancer, due to a faulty gene.

50. Ethical Concerns over Genetic Testing
Genetic testing raises controversial ethical issues.
These include whether a pregnancy should be terminated if a debilitating disease is found in the fetus and whether health insurance companies can deny coverage for individuals with a genetic disease.

51. The Potential of Gene Therapy
Gene therapy is the introduction of a gene into affected cells to replace or augment defective copies of the gene with normal alleles.
In gene therapy, the healthy allele must be sequenced and well understood, and then the DNA has to be introduced in a way that ensures expression of the gene in the correct tissues, in the correct amount, and at the correct time.

52. Introducing Novel Alleles into Human Cells
The current vector of choice in gene therapy are retroviruses.
These are viruses with an RNA genome, including the enzyme reverse transcriptase.
If human genes are packaged into a retrovirus, the virus is capable of inserting the human alleles into a chromosome in a target cell.

54. Using Gene Therapy to Treat X-Linked Immune Deficiency
Gene therapy has been used to treat severe combined immunodeficiency (SCID), a fatal genetic disease whose sufferers have a profoundly weakened immune system.
The type of SCID treated is called SCID-X1, because it is caused by mutations in a gene on the X chromosome.
The gene responsible for SCID-X1 encodes a receptor protein needed for development of immune system cells called T cells.

56. Using Gene Therapy to Treat X-Linked Immune Deficiency
A retrovirus engineered with a normal receptor gene was used to infect cells from bone marrow that produce T cells.
Cells that produced normal receptor protein were then isolated and transferred back into 10 young patients.
Within four months after treatment, nine of the ten patients had normal levels of functioning T cells.
Subsequently, however, four of the boys had developed a type of cancer characterized by unchecked growth of T cells.
Three are responding well to treatment; the fourth has died of cancer.

58. Ethical Concerns over Gene Therapy
Gene therapy is highly experimental, extremely expensive, and intensely controversial.
Although gene therapy holds great promise for the treatment of a wide variety of devastating inherited diseases, fulfilling that promise is almost certain to require many years of additional research and testing, as well as the refinement of legal and ethical guidelines.

59. Biotechnology in Agriculture – Golden Rice
Most strategies for genetic engineering in agriculture focus on one of three objectives:
Reducing herbivore damage.
Making crops more resistant to herbicides.
Improving the quality of food products.

60. Rice as a Target Crop
Although half the world’s population depends on rice as its staple food, this grain contains no vitamin A.
Lack of vitamin A in the diet may cause blindness in children as well as increased susceptibility to disease.
However, rice does contain b-carotene, which is a precursor of vitamin A.
Scientists set out to develop rice enriched in b-carotene.

61. Synthesizing b-Carotene in Rice
The synthetic pathway for b-carotene has three enzymes.
To produce transgenic rice strains capable of producing b-carotene, the three genes that code for these enzymes had to be inserted into rice plants.

63. The Agrobacterium Transformation System
Agrobacterium tumefaciens is a bacterium that infects plants, producing a tumorlike growth called a gall.
A. tumefaciens is often used for genetic transformation of plants through transfer of its Ti (tumor-inducing) plasmid.
A section of the Ti plasmid, called T-DNA, is incorporated into the genome of the host plant cell.
Recombinant genes added to the T-DNA will be expressed in the new host plant.

65. Using the Ti Plasmid to Produce Golden Rice
To develop golden rice, researchers modified Ti plasmids so that they contained the genes for the three enzymes needed to synthesize β-carotene. They then exposed plant embryos to Agrobacterium cells containing these genetically modified Ti plasmids.
A transgenic plant was produced that is now called golden rice, because its high concentration of b-carotene gives it a yellow color.

67. Ethical Concerns
Researchers and consumer advocates have expressed concerns about the increasing numbers and types of genetically modified foods available today.