Analyzing and Engineering Genes

1. Analyzing and Engineering Genes 19

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.

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.

9. 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.

10. 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.

11. 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.

13. 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.

14. Genetic Recombination in Bacteria BLAST Animation: Genetic Recombination in Bacteria

15. 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.

19. 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.

20. 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.

21. 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.

25. Screening a DNA Library • A DNAprobe 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.

27. 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.

31. 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.

32. 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.

33. Producing Human Growth Hormone Web Activity: Producing Human Growth Hormone

34. 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.

35. 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.

39. 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.

43. Polymerase Chain Reaction Web Activity: The Polymerase Chain Reaction

44. 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.

45. 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.

46. 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.

47. 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.