Biotechnology

1. Biotechnology Chapter 17

2. DNA Manipulation The molecular biology revolution started with the discovery of restriction endonucleases -Enzymes that cleave DNA at specific sites These enzymes are significant in two ways 1. Allow a form of physical mapping that was previously impossible 2. Allow the creation of recombinant DNA molecules (from two different sources)

3. DNA Manipulation Restriction enzymes recognize DNA sequences termed restriction sites There are two types of restriction enzymes: -Type I = Cut near the restriction site -Rarely used in DNA manipulation -Type II = Cut at the restriction site -The sites are palindromes -Both strands have same sequence when read 5’ to 3’

4. DNA Manipulation Type II enzymes produce staggered cuts that generate “sticky ends” -Overhanging complementary ends Therefore, fragments cut by the same enzyme can be paired DNA ligase can join the two fragments forming a stable DNA molecule

6. Gel Electrophoresis A technique used to separate DNA fragments by size The gel (agarose or polyacrylamide) is subjected to an electrical field The DNA, which is negatively-charged, migrates towards the positive pole -The larger the DNA fragment, the slower it will move through the gel matrix DNA is visualized using fluorescent dyes

9. Transformation Transformation is the introduction of DNA from an outside source into a cell Natural transformation occurs in many species -However, not in E. coli, which is used routinely in molecular biology labs -Artificial transformation techniques have been developed to introduce foreign DNA into it

10. Molecular Cloning A clone refers to a genetically identical copy Molecular cloning is the isolation of a specific DNA sequence (usually protein-encoding) -Sometimes called gene cloning The most flexible and common host for cloning is E. coli Propagation of DNA in a host cell requires a vector

11. Vectors Plasmids are small, circular extrachromosomal DNA molecules -Used for cloning small pieces of DNA -Have three important components 1.Origin of replication 2.Selectable marker 3.Multiple cloning site (MCS)

12. Vectors

13. Vectors Phage vectors are modified bacterial viruses -Most based on phage lambda (l) of E. coli -Used to clone inserts up to 40 Kbp -Have two features not shared with plasmid vectors -They kill their host cells -They have linear genomes -Middle replaced with inserted DNA

14. Vectors

15. Vectors Artificial chromosomes -Used to clone very large DNA fragments -Bacterial artificial chromosomes (BACs) -Yeast artificial chromosomes (YACs)

16. DNA Libraries A collection of DNA fragments from a specific source that has been inserted into host cells A genomic library represents the entire genome A cDNA library represents only the expressed part of the genome -Complementary DNA (cDNA) is synthesized from isolated mRNA using the enzyme reverse transcriptase

20. DNA Libraries Molecular hybridization is a technique used to identify specific DNAs in complex mixtures -A known single-stranded DNA or RNA is labeled -It is then used as a probe to identify its complement via specific base-pairing -Also termed annealing

21. DNA Libraries Molecular hybridizationis the most common way of identifying a clone in a DNA library -This process involves three steps: 1. Plating the library 2. Replicating the library 3. Screening the library

23. DNA Analysis Restriction maps -Molecular biologists need maps to analyze and compare cloned DNAs -The first maps were restriction maps -Initially, they were created by enzyme digestion & analysis of resulting patterns -Many are now generated by computer searches for cleavage sites

24. DNA Analysis Southern blotting -A sample DNA is digested by restriction enzymes & separated by gel electrophoresis -Gel is transferred (“blotted”) onto a nitrocellulose filter -Then hybridized with a cloned, radioactively-labeled DNA probe -Complementary sequences are revealed by autoradiography

28. DNA Analysis Northern blotting -mRNA is electrophoresed and then blotted onto the filter Western blotting -Proteins are electrophoresed and then blotted onto the filter -Detection requires an antibody that can bind to one protein

29. DNA Analysis RFLP analysis -Restriction fragment length polymorphisms (RFLPs) are generated by point mutations or sequence duplications -These fragments are often not identical in different individuals -Can be detected by Southern blotting

31. DNA Analysis DNA fingerprinting -An identification technique used to detect differences in the DNA of individuals -Makes use of a variety of molecular procedures, including RFLP analysis -First used in a US criminal trial in 1987 -Tommie Lee Andrews was found guilty of rape

32. DNA Analysis

33. DNA Analysis DNA sequencing -A set of nested fragments is generated -End with known base -Separated by high-resolution gel electrophoresis, resulting in a “ladder” -Sequence is read from the bottom up

34. DNA Analysis DNA sequencing -The enzymatic method was developed by Frederick Sanger -Dideoxynucleotides are used as chain terminators in DNA synthesis reactions

36. DNA Analysis DNA sequencing -The enzymatic technique is powerful but is labor intensive and time-consuming -The development of automated techniques made sequencing faster and more practical -Fluorescent dyes are used instead of radioactive labels -Reaction is done in one tube -Data are assembled by a computer

38. DNA Analysis Polymerase chain reaction (PCR) -Developed by Kary Mullis -Allows the amplification of a small DNA fragment using primers that flank the region -Each PCR cycle involves three steps: 1. Denaturation (high temperature) 2. Annealing of primers (low temperature) 3. DNA synthesis (intermediate temperature) -Taq polymerase

39. After 20 cycles, a single fragment produces over one million (220) copies!

40. After 20 cycles, a single fragment produces over one million (220) copies!(Cont.)

41. DNA Analysis Polymerase chain reaction (PCR) -Has revolutionized science and medicine because it allows the investigation of minute samples of DNA -Forensics -Detection of genetic defects in embryos -Analysis of mitochondrial DNA from early human species

42. DNA Analysis Yeast two-hybrid system -Used to study protein-protein interactions -Gal4 is a transcriptional activator with a modular structure -The Gal4 gene is split into two vectors -Bait vector: has DNA-binding domain -Prey vector: has transcription-activating domain -Neither of these alone can activate transcription

43. DNA Analysis Yeast two-hybrid system -When other genes are inserted into these vectors, they produce fusion proteins -Contain part of Gal4 and the protein of interest -If the proteins being tested interact, Gal4 function will be restored -A reporter gene will be expressed -Detected by an enzyme assay

46. Genetic Engineering Has generated excitement and controversy Expression vectors contain the sequences necessary to express inserted DNA in a specific cell type Transgenic animals contain genes that have been inserted without the use of conventional breeding

47. Genetic Engineering In vitro mutagenesis -Ability to create mutations at any site in a cloned gene -Has been used to produce knockout mice, in which a known gene is inactivated -The effect of loss of this function is then assessed on the entire organism -An example of reverse genetics