1. Genetics: Analysis and Principles Robert J. Brooker CHAPTER 18 RECOMBINANT DNA TECHNOLOGY

2. Cloning experiments usually involve two kinds of DNA molecules Chromosomal DNA or cDNA Serves as the source of the DNA segment of interest Vector DNA Serves as the carrier of the DNA segment that is to be cloned Cloning Experiments Involve Chromosomal and Vector DNA

3. The cell that harbors the vector is called the host cell When a vector is replicated inside a host cell, the DNA that it carries is also replicated The vectors commonly used in gene cloning were originally derived from two natural sources 1. Plasmids 2. Viruses Commercially available plasmids have selectable markers Typically, genes conferring antibiotic resistance to the host cell Table 18.2 provides a general description of several vectors used to clone small segments of DNA

4. Insertion of chromosomal DNA into a vector requires the cutting and pasting of DNA fragments The enzymes used to cut DNA are known as restriction endonucleases or restriction enzymes These bind to specific DNA sequences and then cleave the DNA at two defined locations, one on each strand Figure 18.1 shows the action of a restriction endonuclease Cloning Experiments Involve Enzymes that Cut and Paste DNA

5. Figure 18.1 To be continued

7. Restriction enzymes bind to specific DNA sequences These are typically palindromic For example, the EcoRI recognition sequence is 5’ GAATTC 3’ 3’ CTTAAG 5’ Some restriction enzymes digest DNA into fragments with “sticky ends” Other restriction enzymes generate blunt ends

9. This interaction is not stable because it involves only a few hydrogen bonds Add DNA ligase which covalently links the DNA backbones Figure 18.1 To establish a permanent connection, the sugar-phosphate backbones of the two DNA fragments must be covalently linked A recombinant DNA molecule

10. Step 1. The chosen piece of DNA is ‘cut’ from the source organism using restriction enzymes. Step 2. The piece of DNA is ‘pasted’ into a vector and the ends of the DNA are joined with the vector DNA by ligation.ligation Step 3. The vector is introduced into a host cell, often a bacterium or yeast, by a process called transformation. The host cells copy the vector DNA along with their own DNA, creating multiple copies of the inserted DNA.hostcellyeasttransformation Step 4. The vector DNA is isolated (or separated) from the host cells’ DNA and purified. DNA that has been ‘cut’ and ‘pasted’ from an organism into a vector is called recombinant DNA. Because of this, DNA cloning is also called recombinant DNA technology. The Steps in Gene Cloning

11. The procedure shown seeks to clone the human  -globin gene into a plasmid vector The vector carries two important genes amp R  Confers antibiotic resistance to the host cell lacZ  Encodes  -galactosidase Provides a means by which bacteria that have picked up the cloned gene can be identified

13. Figure 18.2 Note: In this case, the  -globin gene was inserted into the plasmid It is also possible for any other DNA fragment to be inserted into the plasmid And it is possible for the plasmid to circularize without an insert This is called a recircularized vector This is termed a hybrid vector

14. Figure 18.2 Cells that are able to take up DNA are called competent cells This step of the procedure is termed transformation.

15. Nonrecombinant: recircularized Recombinant: vector plus inserted cloned gene Selection for vector: ampicillin resistance Selection for recombinant vs. nonrecombinant vector:  -galactosidase activity Selection for gene of interest?

16. The growth media contains two relevant compounds: IPTG (isopropyl-  -D-thiogalactopyranoside) A lactose analogue that can induce the lacZ gene X-Gal (5-bromo-4-chloro-3-indoyl-  -D-galactoside) A colorless compound that is cleaved by  -galactosidase into a blue dye The color of bacterial colonies will therefore depend on whether or not the  -galactosidase is functional If it is, the colonies will be blue If not, the colonies will be white In this experiment Bacterial colonies with recircularized vectors form blue colonies While those with hybrid vectors form white colonies

17. The net result of gene cloning is to produce an enormous amount of copies of a gene During transformation, a single bacterial cell usually takes up a single copy of the hybrid vector Amplification of the gene occurs in two ways: 1. The vector gets replicated by the host cell many times 2. The bacterial cell divides approximately every 30 minutes

18. To clone DNA, one can start with a sample of RNA The enzyme reverse transcriptase is used Uses RNA as a template to make a complementary strand of DNA DNA that is made from RNA is called complementary DNA (cDNA) It could be single- or double-stranded Synthesis of cDNA is presented in Figure 18.4 cDNA

19. Figure 18.4 polyA tail

20. From a research perspective, an important advantage of cDNA is that it lacks introns This has two ramifications 1. It allows researchers to focus their attention on the coding sequence of a gene 2. It allows the expression of the encoded protein Especially, in cells that would not splice out the introns properly (e.g., a bacterial cell)

21. Gel electrophoresis Nucleic acid electrophoresis separates DNA and RNA fragments by size smaller fragments migrate at a faster rate through a gel than large fragments.

23. Another way to copy DNA is a technique called polymerase chain reaction (PCR) It was developed by Kary Mullis in 1985 Unlike gene cloning, PCR can copy DNA without the aid of vectors and host cells The PCR method is outlined in Figure 18.6 Polymerase Chain Reaction

24. The starting material for PCR includes 1. Template DNA Contains the region that needs to be amplified 2. Oligonucleotide primers Complementary to sequences at the ends of the DNA fragment to be amplified These are synthetic and about 15-20 nucleotides long 3. Deoxynucleoside triphosphates (dNTPs) Provide the precursors for DNA synthesis 4. Taq polymerase DNA polymerase isolated from the bacterium Thermus aquaticus This thermostable enzyme is necessary because PCR involves heating steps that inactivate most other DNA polymerases Refer to Figure 18.6

25. The polymerase chain reaction (PCR)

26. PCR is carried out in a thermocycler, which automates the timing of each cycle All the ingredients are placed in one tube The experimenter sets the machine to operate within a defined temperature range and number of cycles Figure 18.6 Binding of the primers to the DNA is called annealing

27. Figure 18.6 With each successive cycle the relative amount of this type of DNA fragment increases. Therefore, after many cycles, the vast majority of DNA fragments only contain the region that is flanked by the two primers The sequential process of denaturing-annealing- synthesis is then repeated for many cycles A typical PCR run is likely to involve 20 to 30 cycles of replication This takes a few hours to complete After 20 cycles, a DNA sample will increase 2 20 -fold (~ 1 million-fold) After 30 cycles, a DNA sample will increase 2 30 -fold (~ 1 billion-fold)

28. 18.2 DETECTION OF GENES AND GENE PRODUCTS Molecular geneticists usually want to study particular genes within the chromosomes of living species This presents a problem, because chromosomal DNA contains thousands of different genes The term gene detection refers to methods that distinguish one particular gene from a mixture of thousands of genes Scientists have also developed techniques to identify gene products RNA that is transcribed from a particular gene Protein that is encoded in an mRNA