BIOL 1107 SI - Biotechnology PDF

Biotechnology Please Sign In! 10/24/24 BIOL 1107 Supplemental Instruction Shyam Nambiar DNA Tech This chapter is all about techniques done in the lab ○ These are not processes that are happening in an organism! DNA Technology - techn...

Biotechnology Please Sign In! 10/24/24 BIOL 1107 Supplemental Instruction Shyam Nambiar DNA Tech This chapter is all about techniques done in the lab ○ These are not processes that are happening in an organism! DNA Technology - techniques to isolate, purify, analyze, and manipulate DNA sequences ○ Use of DNA Tech is called genetic engineering Genetically modified organism (GMO) - a genetically altered organism whose genome has been engineered to introduce or change a genetically controlled trait Biotechnology - any technique used to make or modify products or processes of a biological system or living organism ○ Includes genomics (characterization of whole genomes and bioinformatics (math and computer science applied to biological data)  Gene Cloning Gene cloning - the method of producing many identical copies of a gene of interest in a host cell Cloning Vectors - DNA molecules into which a DNA fragment is inserted to form a recombinant DNA molecule for the purpose of cloning → ex) bacterial plasmid ○ 2 bacterial plasmids (one that will contain recombinant DNA and one control) are isolated that contain the LacZ gene and ampR gene (antibiotic resistance) and a gene of interest is isolated from a cell ○ Restriction endonucleases recognize a specific sequence (restriction sites) of the DNA on the LacZ gene of 1 of the bacterial plasmids and cleave to form sticky ends ○ The gene of interest is cleaved to have sticky ends that are complementary to the plasmid sticky ends ○ The gene of interest is inserted into the plasmid and is sealed with ligase, producing recombinant DNA (fragments of DNA from two or more sources joined together) ○ The DNA recombinant plasmid is placed into one bacteria, while the control is placed into another ○ Recombinant DNA bacteria and controls (bacteria without recombinant DNA plasmid and bacteria without any plasmid at all) are plated in the presence of an antibiotic ○ Recombinant DNA bacteria will appear white because it inhibits the function of the LacZ gene, the bacteria without recombinant DNA will appear blue because it has a functional LacZ gene, and the bacteria without a plasmid will die because it does not have antibiotic resistance PCR Polymerase Chain Reaction (PCR) - In vitro DNA Replication → allows for DNA amplification ○ Allows for the isolation of sequences for amplification rather than the whole DNA molecule ○ PCR cycle runs 20-30 times, producing millions of copies of target sequence Primer - specific sequence that allows for the isolation of the sequence of interest Cycle: ○ Denaturation - Sample containing DNA target sequence, DNA Polymerase, nucleotides, and primers is heated to 95C → dentures DNA to single strands ○ Annealing - sample is cooled to 55-65C → primers anneal to their complementary sequences on either end of the target sequence of DNA ○ Extension - sample is heated to 72C (ideal temp for DNA Pol) → target sequence is replicated producing 2 identical copies of DNA Gel Electrophoresis Gel electrophoresis - DNA, RNA, or other protein molecules are loaded into wells in a gel that is subjected to an electrical field → the molecule separates by size, charge, or other properties depending on the experimental design Agarose gel electrophoresis - used to compare PCR results → size of the amplified DNA is compared to a standard DNA marker ladder to see how far the amplified DNA band travels in relation to the ladder. cDNA Central Dogma of Biology: DNA → RNA → Protein Reverse transcriptase - the only enzyme that disobeys the central dogma ○ Can synthesize DNA from a sequence of mRNA ○ DNA synthesized in this manner is called complementary DNA (cDNA) ○ mRNA is isolated from a cell mRNA has a Poly-A tail at its end ○ Primer of T DNA nucleotides is made (complementary to the Poly-A tail) ○ Reverse transcriptase synthesizes DNA complementary to the mRNA strand ○ RNase enzyme degrades the mRNA strand and DNA Pol synthesized a DNA strand in its place ○ Results in cDNA Genetic Engineering Expression vector - a cloning vector that has regulatory sequences that allow for transcription and translation of a gene Transgenic - organisms that have been engineered to alter their genes Gene targeting - knocking out, replacing, or adding genes into a genome Stem cells - undifferentiated cells that proliferate indefinitely and has the ability to become a precursor cell that will eventually differentiate into a specific type of cell ○ Adult stem cells function to replace specialized cells of tissues and are multipotent, meaning they are limited in the type of cells they can give rise to (ie. hematopoietic stem cells can only differentiate into blood cells) ○ Embryonic stem cells - only found in early-stage embryos and are pluripotent, meaning they have the ability to differentiate into any cell line Knockout mouse - embryonic stem cells of mice are extracted, introduced to transgenes that code for the nonfunction of a target gene, then injected back into early stage embryonic mice ○ Embryonic stem cells with transgene are differentiated into tissues of the growing mouse ○ Mice with the transgenic genes are bred with other mice who were injected ○ Offspring that are homozygous recessive for the transgene are knockout mice CRISPR-Cas9 CRISPR - Clustered Regularly Interspaced Short Palindromic Repeats Cas9 - DNA Endonuclease CRISPR-Cas9 protein contains guide RNA that is complementary to the target gene ○ Once recognized, the Cas9 endonuclease cleaves the target gene and a new gene is able to be inserted or resynthesized by DNA Pol Cas9 must recognize PAM (Protospacer Adjacent Motif) sequences associated with the gene in order to cleave https://www.youtube.com/watch?v=2pp17E4E- O8&ab_channel=McGovernInstitute Gene Therapy Gene Therapy - introduction of a healthy gene into a cell line to correct a genetic disorder Germline gene therapy - when the gene is introduced to the germline cells ○ Not allowed in humans Somatic gene therapy - adult body cells are cultured and transformed with an expression vector containing target transgene and reintroduced to the body ○ Used in humans Sickle-Cell Mutation Sickle cell mutation is caused by a single base pair mutation ○ Because of this mutation, the restriction sites on the b-globin gene are altered ○ In a normal gene, there are two sites for fragmentation ○ In the mutated gene, there is only one Restriction fragment length polymorphisms (RFLPs) - restriction enzyme generated DNA-fragments of different lengths from the same region of the genome ○ Typically analyzed using agarose gel electrophoresis Single-Nucleotide Polymorphism (SNP) - single base-pair mutation ○ ex) sickle cell mutation DNA Fingerprinting DNA Profiling - technique used to identify individuals of the same species by examining their unique DNA sequences ○ Commonly used for forensics and paternity testing PCR is used to analyze specific DNA sequence variations at specific loci in the genome ○ After PCR is performed, the results are analyzed in gel electrophoresis Short Tandem Repeats (STRs) - is s short sequence of DNA repeated in a sequence (2-6 bps long), the number of repeats at each loci varies among individuals in a population DNA that is produced from RNA is called A) mRNA B) cDNA C) tRNA D) None of the above DNA that is produced from RNA is called A) mRNA B) cDNA C) tRNA D) None of the above If a researcher wanted to amplify a specific gene sequence, they would use A) CRISPR Cas 9 B) Polymerase Chain Reaction C) Germline gene therapy D) DNA fingerprinting If a researcher wanted to amplify a specific gene sequence, they would use A) CRISPR Cas 9 B) Polymerase Chain Reaction C) Germline gene therapy D) DNA fingerprinting What must CRISPR Cas 9 recognize in order to cleave the target DNA and replace it with a new sequence A) Guide RNA B) Protospacer Adjacent Motif C) cDNA D) Sticky ends E) A and B What must CRISPR Cas 9 recognize in order to cleave the target DNA and replace it with a new sequence A) Guide RNA B) Protospacer Adjacent Motif C) cDNA D) Sticky ends E) A and B DNA Fingerprinting utilizes _____ to identify individuals A) SNPs B) STRs C) RFLPs D) B and C DNA Fingerprinting utilizes _____ to identify individuals A) SNPs B) STRs C) RFLPs D) B and C Where would CRISPR most effectively be used? A) In embryonic cells B) In stem cells C) In neurons D) In adult cells E) A and B Where would CRISPR most effectively be used? A) In embryonic cells B) In stem cells C) In neurons D) In adult cells E) A and B Announcements Exam 3 is under 2 weeks away (11/4)! My office hours are Wednesday 12:15 pm - 2:15 pm in ROWE 217 (building across from library) Email is [email protected] This slideshow is posted on HuskyCT as a PDF as well as a link to the google drive with this slideshow and other resources. Any Questions?