Introduction to Studying DNA

1. Introduction to Studying DNA Describe the structure and function of DNA and explain how proteins are made Differentiate between eukaryotic and prokaryotic cells and chromosome structure and explain how this difference impacts gene regulation in the two cell types Differentiate between bacterial cultures grown in liquid and solid media and explain how to prepare each type using sterile technique Discuss the characteristics of viruses and their importance in genetic engineering Explain the fundamental process of genetic engineering and give examples of the following applications: recombinant DNA technology, site-specific mutagenesis, and gene therapy Describe the process of gel electrophoresis and discuss how the characteristics of molecules affect their migration through a gel

2. DNA Structure and Function • The manipulation of genetic information, specifically the deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) codes, is the center of most biotech research and development. • The genetic information within cells is stored in DNA • Before we get into DNA lets review Cells!

3. A Review of Cell Structure Online activity on cells: http://learn.genetics.utah.edu/content/begin/cells/ • There are two major types of cells • Prokaryotic Cells • Eukaryotic Cells • Key Terms to Remember! • Plasma Membrane– double-layer structure of lipids and proteins that surrounds the outer surface of cells • Cytoplasm – inner contents of a cell between the nucleus and plasma membrane • Organelles– structures in the cell that perform specific functions

5. Prokaryotic Cells (include bacteria) • No nucleus and no organelles

6. Eukaryotic cells (plant cells, animal cells) • Have a nucleus and many organelles • Organelles • Nucleus • Mitochondria • Endoplasmic reticulum • Golgi apparatus

7. Eukaryotic Cells

11. Other Important molecules in the Cell

12. Types of Cells Used in Biotechnology • Vero cellsare a cell line which is widely used to make vaccines. The cell line was derived from kidney epithelial cells of the African Green Monkey. The cell line was established in 1962 by Japanese scientists. Vero cells are susceptible to a broad range of viruses so they are used to develop vaccines diseases associated with those viruses. One of the vaccines for which Vero cells have been used in recent years is the vaccine against influenza ("the flu"). • HeLa Cells cells are the first immortal cell line, or a cell line that continues to reproduce and "live" outside the human body. These cells have been used in cell research in projects that have benefited mankind around the world. The original cells were taken from cancerous cervical tumor from a poor African-American woman named Henrietta Lacks who died from cervical cancer in 1951.

13. Escherichia coli(commonly abbreviated E. coli) is a gram negative, rod shaped bacteria that is commonly found in the lower intestine of warm blooded organisms. • Most E. coli strains are harmless, but some can cause serious food poisoning in humans, and are occasionally responsible for product recalls. The harmless strains are part of the normal flora of the gut. • E. coli are not always confined to the intestine, and their ability to survive for brief periods outside the body makes them an ideal indicator organism to test environmental samples for fecal contamination. • The bacteria can also be grown easily and its genetics are comparatively simple and easily manipulated or duplicated through a process of metagenetics, making it one of the best-studied prokaryotic model organisms, and an important species in biotechnology and microbiology.

14. Chinese hamster ovary cells or CHOcells are cells that have been derived from the ovary of the Chinese hamster. In year 1957, scientist T. Puck at Dr. George Yerganian's laboratory at the Boston Cancer Research Foundation used a female Chinese hamster to extract this cell line. Today, it is a widely used mammalian cell line in biological research since its introduction in the year 1960. • One of the characteristics of CHO cells is that it requires the amino acid “Proline” for its growth and it’s an adherent monolayer cell line. • A rapid growing cell line with excellent ability to express recombinant protein makes it the cell line of choice in experiments relating to gene expression, genetics, toxicity screening and nutritional studies.

15. Viruses • Viruses are particles of nucleic acid, protein, and in some cases, lipids. • Viruses can reproduce only by infecting living cells. • Viruses differ widely in terms of size and structure. • As different as they are, all viruses have one thing in common: They enter living cells and, once inside, use the machinery of the infected cell to produce more viruses.

16. A virus's protein coat is called its capsid. • The capsid includes proteins that enable a virus to enter a host cell. • The capsid proteins of a typical virus bind to receptors on the surface of a cell and “trick” the cell into allowing it inside. • Once inside, the viral genes are expressed. • The cell transcribes and translates the viral genetic information into viral capsid proteins. • Sometimes that genetic program causes the host cell to make copies of the virus, and in the process the host cell is destroyed

17. Because viruses must bind precisely to proteins on the cell surface and then use a host's genetic system, most • Viruses are highly specific to the cells they infect. • Plant viruses infect plant cells • Most animal viruses infect only certain related species of animals • Bacterial viruses infect only certain types of bacteria. Viruses that infect Bacteria are called bacteriophages. To View how a virus attacks watch this video!

18. DNA Structure Learn the Basics Website: http://learn.genetics.utah.edu/content/begin/tour/ Building block of DNA is the nucleotide • Each nucleotide is composed of • Pentose (5-carbon) sugar called deoxyribose • Phosphate molecule • A nitrogenous base • The nitrogenous bases are the interchangeable component of a nucleotide • Each nucleotide contains one base • Adenine (A), thymine (T), guanine (G) or cytosine (C)

19. Nucleotides are joined together to form long strands of DNA and each DNA molecule consists of two strands that join together and wrap around each other to form a double helix • Nucleotides in a strand are held together by phosphodiester bonds • Each strand has a polarity – a 5’ end and a 3’ end

21. The two strands of a DNA molecule are held together by hydrogen bonds • Formed between complementary base pairs • Adenine (A) pairs with thymine (T) • Guanine (G) pairs with cytosine (C) • The two strands are antiparallel because their polarity is reversed relative to each other

22. Chromosomes • Chromosome Structure • Chromosomes – highly coiled and tightly condensed package of DNA and proteins • Occurs only during DNA replication • Chromatin– strings of DNA and DNA-binding proteins called histones • State of DNA inside the nucleus when the cell is NOT dividing • Most human cells have two sets (pairs) of 23 chromosomes, or 46 chromosomes total • Called homologous pairs • Autosomes – chromosomes 1-22 • Sex chromosomes – chromosome pair # 23 • X and Y chromosomes • Gametes (sex cells) contain a single set of 23 chromosomes (haploid number, n)

24. DNA Replication and Cell Division • DNA Replication • Cells divide by a process called mitosis • Sex cells divide by a slightly different process called meiosis • Mitosis • One cell divides to form two daughter cells, each with an identical copy of the parent cell DNA • In order to accomplish this, the DNA of the parent cell must be copied prior to mitosis • DNA undergoes Semiconservative Replication • Replication occurs in such a manner that, after replication, each helix contains one original (parental) DNA strand and one newly synthesized DNA strand

26. Steps in DNA Replication • Unwinding the DNA • Helicase enzyme breaks the hydrogen bonds holding the two DNA strands together; “unzips” DNA • DNA binding proteins hold the strands apart • Separation of strands occurs in regions called origins of replication • Adding short segments of RNA • Primase enzyme adds RNA primers • RNA primers start the replication process

27. 3. Copying the DNA DNA polymerase enzyme binds to the RNA primers Uses nucleotides to synthesize complementary strands of DNA Always works in one direction – 5’ to 3’ direction

29. RNA and Protein SynthesisOnline activity: http://learn.genetics.utah.edu/content/begin/dna/

30. Transcription • Occurs only in genes • RNA polymeraseunwinds DNA helix and copies one strand of DNA into RNA • Binds to a promotor region • Copies DNA in a 5’ to 3’ direction into RNA • Uses nucleotides • Adenine, uracil, guanine, and • At end of gene, RNA polymerase encounters the termination sequence • RNA polymerase and newly formed strand of RNA are released from DNA molecule

31. RNA strand is called a messenger RNA (mRNA) • Multiple copies of mRNA are transcribed from each gene during transcription • The mRNA then gets processed • Initial mRNA produced is the primary transcript • Immature and not fully functional • A series of modifications before primary transcripts are ready for protein synthesis • RNA splicing • Polyadenylation • Addition of a 5’ cap

33. How Is mRNA read? • mRNA is read during a process called translation • Translation takes place in the cytoplasm • Works in three nucleotide units of mRNA called codons • Each codon codes for a single amino acid • One amino acid may be coded for by more than one codon • Start codon (AUG) • Stop codons

35. There are three different types of RNA involved in the translation process • mRNA – exact copy of the gene; carries the genetic code from nucleus to the cytoplasm • rRNA – component of ribosomes, the organelles responsible for protein synthesis • tRNA – transports amino acids to ribosome

36. Translation of mRNA • 1.Initiation– small ribosome subunit binds to 5’ end of mRNA • Moves along the mRNA until the start codon is found (AUG) • 2.Elongation – tRNAs, carrying the correct amino acid, enter the ribosome, one at a time, as the mRNA code is read • 3.Termination – ribosome encounters the stop codon • Newly formed protein is released

38. Applying what we know to Biotechnology • Many Biotech efforts modify DNA molecules with the goal of affecting protein production • In humans, about 40,000 genes are needed for an organism to function • A typical cell synthesizes more than 2000 different kinds of proteins and hundreds or thousands of copies of these proteins are usually needed at any given time • If you multiply these numbers by the hundreds of types of cells we have in our body, the numbers reach into the millions

39. The entire sum of DNA in a cell is variable from organism to organism, but every cell within an organism, except sex cells, has the same genome. • Even though there are different quantities of DNA in cells from different organisms, the DNA itself is virtually identical.

40. Similarities in DNA Molecules Among Organisms • All DNA molecules are made of A,T,C,G • Virtually all DNA molecules form a double helix • The nucleotides connect to one another via phosphodiester bonds between the sugar and phosphate • Hydrogen bonds hold each base to its complementary base creating the two sides of the DNA molecule • The amount of A is always equal to the amount of T • The amount of G is always equal to the amount of C

41. Bacterial Transformation • In prokaryotic cells, the DNA is floating in the cytoplasm. • It typically contains only one, long, circular DNA molecule (chromosome) • It is usually folded in on itself and only contains several thousand genes (compared to a human who has around 40,000 genes)

42. Some bacteria contain small rings of DNA that are outside of the “chromosome” floating in the cytoplasm called plasmids • Plasmids only contain a few genes (5 to 10) that usually code for proteins that offer some additional characteristics that may be needed only under extreme conditions.

43. The most common type of plasmids are R plasmids • R plasmids contain antibiotic resistance genes • These genes allow the bacteria to survive exposure to antibiotics that would normally kill them • Bacteria can transfer genetic information between themselves by a process called conjugation. • Transferring plasmids give bacteria a way of “evolving” by gaining new and better characteristics

45. Because plasmids are pieces of DNA that can accept, carry, and replicate (clone) other pieces of DNA, they are often used as vectors. • A Vector is a vehicle used to transfer the genetic material such as DNA sequences from the donor organism to the target cell of the recipient organism.

46. How do restriction enzymes work? http://www.phschool.com/science/biology_place/labbench/lab6/enzwork.html • Just like other enzymes, restriction enzymes show specificity for certain substrates (DNA) • They work by cutting the phosphodiester bond (in the sugar-phosphate backbone) that joins adjacent nucleotides in a DNA strand. • The cut DNA within a specific sequence of bases called a recognition sequence or restriction site

48. Isolating and Manipulating DNA • Modifications of DNA can be as simple as changing a single base • Changing DNA sequences may alter the production of some proteins in a cell or organism • New proteins may also be created • Genetic Engineering: the production of rDNA molecules and their insertion into cells • rDNA: (recombinant DNA) :pieces of DNA that have been cut and then pasted back together

49. Recombinant DNA Technology • These technologies are the methods used to create new DNA molecules by piecing together different DNA molecules • When cells accept the rDNA and start expressing the new genes (by making the new proteins) they are considered genetically engineered. • The names of proteins produced in this way are written with an “r” in front of them. • Example : rInsulin

50. Site –Specific mutagenesis • Site-specific mutagenesis refers to the process of inducing changes (mutagenesis) in certain sections (site-specific) of a particular DNA code. • The changes in the DNA code are usually accomplished through the use of chemicals, radiation, or viruses. • Sometimes site-specific mutagenesis is “directed” meaning a scientist is trying to make certain changes in a protein’s structures that will translate into an improved function. • Example: Subtilisin is an enzyme that is added to laundry detergent to remove proteinacous stains (such as blood or gravy) This protein was made from altering the protein subilisin found in fungi. The alteration made the protein able to function in an alkaline soln. such as laundry det.