BIOTECHNOLOGY

1. BIOTECHNOLOGY Campbell and Reece Chapter 20

2. DNA Technology • made possible: • Human Genome Project completed in 9 yrs (2001) • by 2010 genomes of > 7,000 species

3. Recombinant DNA DNA formed when segments of DNA from 2 different species are combined in vitro (in test tube) useful for analyzing genes & gene expression

4. Biotechnology manipulation of organisms or their components to produce useful products includes selective breeding, using bacteria in fermentation

5. Genetic Engineering the direct manipulation of genes for practical purposes

6. DNA Cloning • useful for researcher to have just that portion of DNA working with • 1 gene may be as small as 1/100,000th of a chromosome • bacterial plasmids: circular DNA molecules that replicate separately from bacterial chromosome • used by bacterium when environment changes

7. Gene Cloning

8. Restriction Enzymes used to cut DNA w/in short, specific nucleotide sequences (restriction sites)  set of dbl stranded DNA fragments with single stranded “sticky ends”

9. DNA Ligase sticky ends form H-bonds with sticky ends of C’ bases from other DNA: temporary bonds DNA ligase make bonds permanent: makes covalent bonds in sugar-phosphate backbone

11. Restriction Enzymes Animations http://www.dnalc.org/resources/animations/restriction.html http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/bio37.swf::Restriction%20Endonucleases

12. Cloning Vectors • name given original plasmid • dfn: a DNA molecule that can carry foreign DNA into host cell & replicate there • bacterial plasmids mostly used because • readily available from suppliers • can insert foreign DNA in vitro  bacterial cell • multiply rapidly

13. Cloning Vectors Animation http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_2.html

15. Storing Cloned Genes in DNA Libraries

16. Bacterial Artificial Chromosome (BAC) large plasmids trimmed down so they contain just the genes necessary for replication carry 100 – 300 kb (kilo base pairs), normal plasmid can insert vectors no larger than 10 kb

17. cDNA can make “libraries” using cDNA:

18. Genomic & cDNA Libraries • each have advantages • Genomic library good if: • looking for a gene but not sure where it is in a genome, or what kind of cell to look in • if looking for introns or regulatory sequences as’c w/gene • cDNAlibrarygood if studying: • specific protein • sets of genes expressed in particular cell types • changes in patterns of genes over life of cell (during development of organism)

19. Screening Library for Clones Carrying Gene of Interest Nucleic Acid hybridization: process of base pairing between a gene & a C’ sequence on another nucleic acid molecule C’ molecule = ssDNA or ssRNA = nucleicacid probe probe is made that is C’ to known sequence in gene

20. Nucleic Acid Hybridization

21. Detecting a Specific DNA Sequence by Hybridization with a Nucleic Acid Probe

22. Expressing Cloned Eukaryotic Genes several technical difficulties hinder the expression of cloned eukaryotic genes in bacterial hosts can substitute eukaryotic hosts: yeasts, some insect cell, some mammalian cells that have appropriate expression vectors: a cloning vector that contains a highly active bacterial promoter just upstream of restriction site where eukaryotic gene can be inserted allowing gene to be expressed in bacterial cell or have been genetically engineered to use in specific eukaryotic cells

23. PCR Polymerase Chain Reaction amplifies specific target segment of DNA in vitro using primers that bracket the derived sequence, & a heat-resistant DNA polymerase, & nucleotides

24. PCR

26. What DNA Technology Allows once you have many copies of a gene you can ask questions about its functions, where & when the gene is expressed, or how important is it to the organism

27. Gel Electrophoresis uses a gel made of a polymer (agarose commonly) gel acts like molecular sieve separates nucleic acids or proteins based on charge and size nucleic acids carry (-) charges on phosphate group so  (+) end of gel as move the longer molecules impeded more by “sieve”

28. Gel Electrophoresis

29. Restriction Fragment Analysis fragments produced be restriction enzymes put thru gel electrophoresis  band pattern characteristic of starting molecule & restriction enzyme used able to identify viruses & plasmids by their band patterns then recover DNA from gels 1 way of getting pure samples of DNA – won’t work with DNA from eukaryotic cells: gel electrophoresis  smear not bands

31. RFLP Restriction Fragment Length Polymorphism: single nucleotide polymorphism (SNP) that exists in the restriction site for a particular enzyme, making the site unrecognizable by that enzyme & changing lengths of the restriction fragments formed by digestion with that enzyme found in coding & noncoding DNA

33. Using restriction fragment analysis to distinguish the normal & sickle cell alleles of the human β-globin gene

34. Southern Blotting used to detect certain nucleotide sequences w/in a complex DNA sample compares the restriction fragments produced from different samples of genome DNA

35. Southern Blotting

36. Dideoxy Chain Termination method used to sequence relatively short DNA fragments done by automated sequencing machines

37. Dideoxy Chain Termination • technique: synthesizes a set of DNA strands C’ to original DNA fragment • each strand starts with same primer & ends with dideoxyribonucleotide (ddNTP) • incorporation of ddNTP terminates growing DNA strand because it lacks 3’ –OH group (site of attachment of next nucleotide) • each ddNTP tagged with distinct fluorescent label so identity of nucleotide at end of each strand (ultimately entire strand) is identified

39. Northern Blotting in vitro hybridization with labeled probes looking for specific mRNAs could be used to look at how expression of a gene changes during the embryonic development of organism carry out gel electrophoresis on mRNA

41. RT-PCR • Reverse Transcriptase- Polymerase Chain Reaction • quicker & more sensitive than Northern blotting • isolates mRNA from different developmental stages of organism then add reverse transcriptase to make cDNA which serves as template for PCR amplification using primers from gene being studied • bands will be in samples that originally contained the gene being studied

42. in situ hybridization alternative method used to determine which cells are expressing certain genes done in living organism probes labeled with fluorescent dyes

43. DNA Microarray Assays

44. DNA Microarray Analysis uncover gene interactions suggest correct therapeutic route in cancer treatments

45. in vitro Mutagenesis most common method used to determine function of gene: disable it & observe what happens specific mutations introduced to cloned gene & then mutated gene returned to cell knocking out normal gene in the process

46. RNAi RNA interference method for silencing expression of selected genes uses synthetic dsRNA molecules matching the sequence of gene to trigger breakdown of the gene’s mRNA or to block its translation

47. RNAi important when studying groups of genes to determine how multiple genes interact (basis of systems biology, chap 21) in humans considered unethical to block activity of genes

49. Genome-Wide Association Studies • used to analyze genomes of large #s of humans with certain phenotype or disease • test for genetic markers: DNA sequences that vary in a population • uses SNPs (single nucleotide polymorphisms) • single base pair site where variation is found in at least 1% of population • few million in human genome