I ordered a genetic engineering home lab kit for ~$1900 from the Odin a few months ago. Getting it ready took a while but I received news finally that the order status has been updated to “shipped.” A tracking number was provided, and it’s currently in a state of “pre-shipment,” they’re still awaiting the item. Nevertheless, let’s celebrate the update in this order’s status by preparing for some genetic engineering. In this blog post, I will do an inspectional reading of the book “Principles of Gene Manipulation and Genomics” by S.B. Primrose and R.M. Twyman.
The role of an inspectional reading is to get the most you can out of a book in a limited period of time. For most books it’s to determine the book’s main message, it’s layout and how the structure contributes to the main point. Since I fully intend to do an analytical reading of this book, the inspectional reading will serve to lay the groundwork for it, as well as (80/20 rule) helping me find the sections of the book which will be most immediately relevant to what I want to do. Note that Adler and Doren explained inspectional reading they didn’t necessarily have textbooks in mind. Below are my notes from examining the table of contents, preface, chapter 1 and the final chapter.
Title: "Principles of Gene Manipulation and Genomics" by S.B. Primrose and R.M. Twyman Contents: Book consists of 26 chapters. Outside the first chapter, which is introductory, the other chapters are grouped into parts. Intro chapter 1: "Gene manipulation in the post-genomics era" -- what is gene manipulation? what is it's historical context (including the inventions that make it possible) and what is it for? -- Also includes outline for the rest of the book. This is the section where the authors give tips on how to best read their book. Part I (chapters 2 - 9) Fundamental Techniques of Gene Manipulation Part II (chapters 10 - 15) Manipulating DNA in Microbes, Plants and Animals - Many of these chapters deal with specific challenges unique to the kingdom of life you want to modify Part III (chapters 16 - 24) Genome Analysis, Genomics and Beyond. Part IV (chapters 25 - 26) Application of Gene Manipulation and Genomics Next we'll examine the table of contents at the level of the chapter. As you can see, compared to most books, the table of contents in textbooks goes into great detail. Part I (chapters 2 - 9) Fundamental Techniques of Gene Manipulation Ch. 2 - Basic Techniques. - Overview of techniques necessary for gene manipulation. - Covers Gel Electrophoresis, Blotting (Southern, Northern and Western), PCR, Electroporation Ch. 3 - Cutting and Joining DNA Molecules. - Cutting DNA molucules lists several points about restriction endonucleases and contributing factors which may enable or impede their function in cutting DNA. - Cites DNA Ligase as key for joining molucules "in vitro" - Talks about matching the correct ends for ligase to join and techniques for that. - Also cites alternative (but evidently less preferred) method of joining DNA. Ch. 4 - Basic biology of plasmid and phage vectors - (from my training in biology a plasmid is a circular loop of DNA used by bacteria, and favored by scientists because it's easy to manipulate and insert into a cell, particularly since bacteria don't have a nucleus. A phage is a virus. A phage vector is a virus used to carry (instead of it's own DNA) the mutant DNA that you've prepared for the cell) - Lists several points to consider about plasmids, such as "Plasmids with similar replication and partitioning systems cannot be maintained in the same cell" - Lists several points about bacteriophage lambda after talking about it's "genetic organization." - Talks about single-stranded DNA vectors and use cases for them. Ch. 5 - Cosmids, Phasmids and Other advanced vectors - "Cosmids are plasmids that can be packaged inside bacteriophage lambda." - Lists vectors capable Title: "Principles of Gene Manipulation and Genomics" by S.B. Primrose and R.M. Twymanof handling much larger strands of DNA - Vectors for single stranded dna, for promoting the solubility of expressed proteins, and for maximizing synthesis - The Gateway(R) system for loading DNA into different vectors. Ch. 6 Gene Cloning Strategies (all seem about sequencing DNA and building libraries) - Talks about building a genomic DNA library as well as PCR - Complimentary DNA libraries and PCR as an alternative Ch. 7 DNA Sequencing - Sanger method, pyrosequencing and more. Ch. 8 Changing genes: Site-Directed mutagenesis and protein engineering - Primer-extension or single-primer method, as well as its limitations - Alternatives to primer extension and also PCR for site-directed mutagenesis - Gene shuffling for protein engineering - Random mutations throughout a gene Ch. 9 Bioinformatics - Intro to Nucleotide and protein databases - Analysis by sequence alignment - Algorithms for identifying key regions and non-coding regions in DNA, and analysis from scratch (not by comparing to other known DNA) Part II (chapters 10 - 15) Manipulating DNA in Microbes, Plants and Animals Ch. 10 Cloning in bacteria other than E. Coli - Gram negative vs Gram positive bacteria - Cloning in Archaea Ch. 11 Cloning in Saccharomyces cerevisiae and other fungi - Reasons for S. Cerevisiae, different promoters, special vectors, etc. - Yeast artificial chromosomes can be used to clone and manipulate large quantities of DNA. Ch. 12 Gene transfer to animal cells - Four main strategies: Calcium phosphate + other chemical methods - Physical techniques like electroporation or piercing the cell membrane - Plasmid vectors - Bacterial-derived viruses Ch. 13 Genetic manipulation of animals (generally refers to the entire animal + germline) - Pronuclear microinjection into fertilized mouse eggs - Viruses on early embryos - Embryonic stem cells for gene targeting - Applications of genetically modified mice and gene targeting - Sperm injection for other mammals and birds. - Nuclear transfer to clone animals - gene transfer to xenopus (an aquatic frog) - Gene transfer to fish generally done by microinjection, but new methods are on their way - Gene transfer to fruit flies done by microinjection of the "pole" plasma. Ch. 14 Gene transfer to plants - Using callus cultures to get undifferentiated plant cells. (same topics also discussed in "The Biotechnology of Cannabis Sativa" <link>) - Four main strategies for gene transfer, 1 = mediated by Agrobacterium, which stimulates tumors - Direct DNA transfer by means of particle bombardment and other stuff - Gene Targeting by means of viral vectors in plants Ch. 15 Advanced transgenic technology - Inducible expression (turn the gene on/off via a switch!) - Methods for recombinant DNA and protein-level DNA inhibition. (this book was likely written before CrispR, CAs9) Part III (chapters 16 - 24) Genome Analysis, Genomics and Beyond. - 16. Organization and structure of genomes - Genomes vary by over 5 orders of magnitude. (Viruses & Bacteria, Mitochondria & Organelle DNA, Chloroplast DNA etc.) - Organization of Nuclear DNA in Eukaryotes -Ends with summary of their structural elements - 17. Mapping and Sequencing genomes - (Talks about different strategies and limitations for mapping and sequencing genomes) - 18. Comparative genomics - Broken down into the comparative genomics of bacteria, organelles and eukaryotes - 19. Large Scale Mutagenesis - (about knocking out genes by either an insertion, point mutations or deletions) Transcriptomics and 3 Chapters on Proteomics Part IV (chapters 25 - 26) Application of Gene Manipulation and Genomics - 25. Applications of Genomics: understanding the basis of polygenic disorders and identifying quantitative trait loci. - Talks about linkage (co-inheritance) studies to identify genes correlated to a disease. (we discussed Genome Wide Association Studies briefly in the most recent post on immunology) - Uses inbreeding and responses to drugs as examples/case-studies. - 26. Applications of recombinant DNA technology - Talks about the present and future use of this technology in producing valuable molecules, in improving traits in agriculture (think Norman Borlaug saving a billion lives), and it's use in preventing and curing diseases. Preface: - First edition of the book 25 years ago was "Principles of Gene Manipulation." "Principles of Genome Analysis" was a separate book. Both were combined in this book as the methods and techniques for genetics and genomics now signifantly overlap. - The first part of the book is devoted to E. Coli, as it is the most thoroughly studied and therefore easiest for biologists to work with. It is often also used as a prerequisite for engineering genes in other organisms. - The second part of this book applies the techniques of Part 1 to other organisms and should be understandable to anyone who followed part 1. - Historical information is included to highlight the origins of modern techniques, and to explain techniques that may still be referenced in key papers. - Suggested papers and reviews are provided at the end of each chapter. Chapter 1. - Explains recombinant DNA, the origins of genetics and what genes are. - Gives another outline of the book: - provides an excellent summary of the information in the table of contents - includes charts detailing the roadmap to understanding the information in each part of the book. (for instance for part I): Ch. 2 Basic Techniques --> Ch. 3 Cutting and Joining DNA (so that the DNA can be incorporated into or excised from the genome), Ch. 4 & 5 on Vectors, Ch 6 puts it all together and Ch. 7-9 refer to analysing and tinkering with cloned genes. In this case the final chapter is not a summary chapter, and therefore does not contribute to communicating the main idea in broad strokes.
To conclude, this is a HOWTO book containing the information necessary to tamper with genes and genomes. Part 1 alone is sufficient to perform gene manipulation. Only select chapters in part 2 are needed depending on what species you want to tinker with (is it a plant, mushroom or human?). Part 3 goes into the various -omics as a skill to augment the effectiveness of gene manipulation, and because knocking out genes is frequently used to study their function in a genome, and part 4 stimulates your imagination for how your new-found skills can be used, by demonstrating the various was they are being used right now.
New Goal: finish part 1 by the time my equipment arrives!
“Principles of Gene Manipulation and Genomics 7th Edition” by Sandy B. Primrose and Richard Twyman = http://www.khuisf.ac.ir/prof/images/Uploaded_files/ebooksclub.org__Principles_of_Gene_Manipulation_and_Genomics_Seventh_Edition%5B4993062%5D.PDF
“How to Read A Book, Revised Edition” by Mortimer Adler and Charles van Doren. (Older edition can be found here = http://mathscinotes.com/wp-content/uploads/2016/02/Adler-Mortimer-How-To-Read-A-Book.pdf)