DNA Microarray Assays

1. DNA Microarray Assays • Technique used to study how genes act together to produce & maintain a functioning organism • Which genes are transcribed in different situations such as different tissues or at different stages of development • Consists of tiny amounts of a large number of single strand-stranded DNA fragments representing different genes fixed to a glass slide in a tightly spaced array (grid) • Aka: DNA chip

2. Microarray Technique • Isolate mRNA • Make cDNA by reverse transcription, using fluorescently labeled nucleotides • Apply the cDNA mixture to a microarray; the cDNA will hybridize with any complementary DNA on the microarray • Rinse off excess cDNA & scan for fluorescence • each fluorescent spot represents a gene being expressed • The intensity of fluorescence indicates level of expression

3. Tissue sample Isolate mRNA. LE 20-14 mRNA molecules Make cDNA by reverse transcription, using fluorescently labeled nucleotides. Apply the cDNA mixture to a microarray, a microscope slide on which copies of single-stranded DNA fragments from the organism’s genes are fixed, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. Labeled cDNA molecules (single strands) DNA microarray Rinse off excess cDNA; scan microarray for fluorescent. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample. Size of an actual DNA microarray with all the genes of yeast (6,400 spots)

4. Practical Applications of DNA Microarrays • Track gene expression changes from initial development throughout an organisms lifetime • Contribute to better understanding of certain diseases & suggest new diagnostic techniques or therapies

6. Gene Therapy • Alteration of an afflicted individual’s genes • Experimental: in theory,a normal allele of a defective gene could be inserted into the somatic cells of the tissue affected by the disorder • Somatic cells must multiply throughout patients life (ex: bone marrow cells)

7. Cloned gene Insert RNA version of normal allele into retrovirus. LE 20-16 Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Retrovirus capsid Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient Bone marrow Inject engineered cells into patient.

9. Practical Applications of DNA Technology: Medicine / Pharmaceutical 1)human gene therapy 2)pharmaceutical products -insulin, growth hormone, TPA (dissolves blood clots), proteins that mimic cell surface receptors for viruses like HIV 3) diagnosis of disease

11. Applications of DNA Technology… • Forensic uses • DNA fingerprints: paternity, criminal cases  Environmental uses: microorganisms engineered to break down sewage, oil spills, etc.

12. Applications of DNA Technology…  Agricultural uses (“Pharm” Animals and Plants) 1)livestock -bGH (bovine growth hormone) to enhance milk production -sheep that carry a gene for human blood protein -genes that cause devp’t of larger muscles in cattle -salmon that grow larger due to a modified growth hormone 2) genetically engineered plants - insecticide/herbicide resistance - decaying of ripening/resistance to spoiling - transgenic rice that contain beta-carotene to solve the vitamin A deficiency in poorer countries

13. Regular Tomato Flavor Savr Tomato

14. Agrobacterium tumefaciens Ti plasmid LE 20-19 Site where restriction enzyme cuts T DNA DNA with the gene of interest Recombinant Ti plasmid Plant with new trait

15. Why is this a restriction enzyme site? • Restriction enzymes bind to special hydrogen bond sites • Restriction enzymes cut at GAATTC • Restriction enzymes cut at CTTAAG • Restriction enzymes recognize specific reverse order sequences

16. What are “sticky ends?” • Pieces of DNA that are run through a gel in order to give a unique banding pattern • Single-stranded DNA ends that are available to hydrogen bond to a complimentary single strand • DNA bases that are added to a PCR machine so that multiple exact copies of a DNA sequence can be produced • Segments of DNA that act as a probe in order to diagnose a genetic condition

17. What is this called? plasmid Human gene • RFLP (restriction length polymorphism) • PCR (polymerase chain reaction) • Clone • Recombinant DNA

18. What is the purpose of PCR? • To make billions of identical copies of a specific DNA segment • To separate out DNA segments by size • To help make medical products such as insulin • To help cure diseases such as Cystic Fibrosis

19. What is the basis for DNA movement in gel electrophoresis? • DNA has a (+) charge so it moves towards a (-) electrical terminal • DNA has a (+) charge so it moves towards a (+) electrical terminal • DNA has a (-) charge so it moves towards a (+) electrical terminal • DNA has a (-) charge so it moves towards a (-) electrical terminal

20. Which of the following is NOT an application of biotechnology? • Agricultural products such as bovine growth hormone • Pharmaceutical products such as insulin • Environmental uses; cleaning up oil spills • Medical uses; to help parents have children with specific traits • Medical uses; to help diagnose some diseases

21. Vocabulary terms Transcription is the process of synthesizing RNA using a DNA duplex as template. Translation is the process of synthesizing a protein using an mRNA molecule as a guide. Gene expression refers to the transcription and translation of a gene or set of genes. Gene regulation refers to the control of gene expression.

22. more vocab… Hybridization is the process by which two complementary strands of nucleic acid base pair to one another to form a duplex. If two strands of nucleic acid are not complementary, they will not hybridize to form a duplex. Gene knockouts are experiments in which a gene is deleted from the genome of an organism. Knockouts are used to gain information about the function of a gene.

23. still more vocab… Transcriptional regulation is the control of gene expression at the level of transcription. RNA interference (RNAi) is the phenomenon in which experimentally introduced double-stranded RNA leads to loss of expression of the corresponding cellular gene.