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 2011 Pearson Education Inc 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 2011 Pearson Education Inc Introduction Manipulation of DNA sequences in organisms is known as genetic engineering and techniques used to engineer genes are called recombinant DNA technology 2011 Pearson Education Inc 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 2011 Pearson Education Inc 2011 Pearson Education Inc 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 2011 Pearson Education Inc 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 2011 Pearson Education Inc 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 2011 Pearson Education Inc 2011 Pearson Education Inc 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 2011 Pearson Education Inc 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 2011 Pearson Education Inc 2011 Pearson Education Inc 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 2011 Pearson Education Inc 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 2011 Pearson Education Inc 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 2011 Pearson Education Inc 2011 Pearson Education Inc 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 2011 Pearson Education Inc 2011 Pearson Education Inc 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 2011 Pearson Education Inc 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 2011 Pearson Education Inc 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 2011 Pearson Education Inc Requirements of PCR PCR is