Applied Genomic Technologies

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Recombinant DNA Techniques

Lecture 13 – Barcoding, Genetic Diversity, and Polymorphisms  Universal Primers o Universal Primers are primers that are designed to areas in genes that are highly conserved among different species o Thus universal primers are not species specific o This means that the primers are probably not totally complementary to the sequence in your insect sample  The sequence in the CO1 gene in between where the primers hybridize is less well conserved and thus can be used to differentiate members of different species  To allow for this, we anneal the primers at a lower temperature o Because the primers are not going to be totally complementary to many of the organisms we want to barcode, we do the annealing step of the PCR reaction at 45°C  Because of the low annealing temperature, we do often get non-specific products o We will then send the purified PCR product for sequencing o If you get no product or multiple products, you’ll repeat the PCR under different conditions to try to get a single PCR product, which can be sent for sequencing  CO1 Gene and Barcoding o The cytochrome C oxidase (CO1) gene has been designated the standard gene for barcoding of animals o It is a mitochondrial protein coding gene of about 1500 bp with no introns o The CO1 region that is used for barcoding includes the first half of the gene and is approximately 650 bp long  PCR is used to amplify this region; the PCR product is then sent for sequencing  Mitochondrial DNA o Circular DNA – 17,000 bp o Hundreds of copies per cell o Inherited from mother only – so it’s haploid o No recombination o Contains 37 genes – no introns  22 tRNAs  2 rRNAs  13 protein coding genes involved in cellular respiration o Looks a lot like bacterial DNA!  1) Mitochondrial Genes are Haploid o In doing barcoding, we want to make the sequencing part as easy as possible –we want to be able to isolate DNA, amplify it, and sequence it without having to clone the DNA o Thus it is important to use a haploid gene as our identifying gene  If you used a diploid gene, you would get products with possibly 2 different sequences o So we can’t use nuclear genes – instead we use a mitochondrial gene for animal barcoding  2) Mitochondrial Genes are Present in High Copy Numbers in Cell o Makes it more likely to find good copies  3) Mitochondrial Genes Show Differences Between Species, but Not Much Within Species o Sequence differences among closely related animal species average 5-to 10-fold higher in mitochondrial than nuclear genes o Intraspecific variation in mitochondrial DNA is low in most animal species o Thus small intra specific and large inter specific differences make distinguishing genetic boundaries between species easier, enabling precise identification  Bioinformatics o After we get our sequences, we will use BLAST to try determine the genus and species of your samples  We will also compare the sequence to all the sequences in another database called BOLD, which is a database specific for barcode sequences o We will consider any hit with an identity of 97% or higher the same species o If the match is less than 97%, it means the barcode sequence for the organism is not in GenBank or BOLD o Once we have a set of sequences from the same organisms (i.e., same genus and species) we will also look at genetic diversity within that organism (SNPs) o Finally, if the sequences are of high quality, we will submit them to the BOLD database, and from there they will be imported into GenBank  Organism Classification o Domain, Kingdom, Phylum, Class, Order, Family, Genus, Species

BIMM | 101 | Recombinant DNA Techniques | lecture notes | Lecture 13 – Barcoding  Geneti

Midterm_Rubric.pdf-BENG 183 Midterm November 6 th

Discussion 1  Models of DNA Replication o Conservative Model : Child is composed of 2 brand new strands o Semi-Conservative Model : Child is composed of 1 old strand and 1 new strand o Scattered Model : Random mix of old and new DNA in each strand  DNA Replication Terms (and Proteins) o Helicase : Unwinds and unzips DNA (located right on the fork) o Topoisomerase : Prevents DNA from having too much torque by making little slits (so it’s an endonuclease) (located on the still-zipped part) o Leading Strand : The strand that is created in the direction of the replication fork o Lagging Strand : The strand that is created in the opposite direction (use Okazaki Fragments)  DNA Shortening (and prevention of) o Each time DNA is replicated, it would shorten because the lagging strand runs out of room for primer to latch onto, so the end wouldn’t get replicated o Because of this, we have Telomeres to act as “dummy data” o Telomeres are added by Telomerase (a Reverse Transcriptase)  DNA Repair o Mismatch Excision Repair  Incorrect pairing of nucleotides o Base Excision Repair  Chemical reaction changed a nucleotide o Nucleotide Excision Repair  Messed up DNA structure o End-Joining  Error prone because it takes two ends of DNA that are (probably) unrelated, cuts off any excess so they line up, and sticks them together  DNA Sequencing (Sanger) o Slice up DNA into small pieces, use Primer to start, and use dNTP to elongate new chains and ddNTPs to end elongation o Result will be many different length strands o We put all these into gel electrophoresis (on the negative end) and small pieces travel furthest o Result is 5’ to 3’ sequence from positive to negative  Noncoding RNAs o rRNA : Ribosomal RNA, used for Protein Synthesis o tRNA : Transfer RNA, used for Protein Synthesis o snRNA : Small Nucleolar RNAs, used for mRNA Splicing o Micro RNAs : Gene Silencing (attaching to mRNA) o Xist : Silence X Chromosome (used in females) o Telomerase RNA : Template for addition of telomeres  Solitary vs. Duplicated Genes o Solitary Gene : Found once in a genome o Duplicated Gene : Found multiple times in a genome  Note that they’re not necessarily identical sequences, but they do essentially the same thing (create a similar protein)  PCR vs. DNA Replication o Both are some form of replicating DNA, but PCR is done in the lab and DNA Replication is natural o Each cycle of PCR doubles the number of DNA strands  After n cycles of PCR, we will have 2 n strands  Extra DNA o Not all DNA is actually really used (actually very small portion is) o People still don’t really know why

BIMM | 100 | Molecular Biology | lecture notes | Discussion 1  Models of DNA R

o What do the BLAST results tell you? Be able to interpret BLAST results! (Given different % identities, are two organisms same species or genus?) o What do ClustalW results tell you? Be able to interpret ClustalW results! SNPs (p 276-278 in Genes to Genomes 3 rd ed) o What are SNPs? o Synonymous versus non-synonymous SNPs: how can you tell? (translate) o What are haplotypes? o PTC taster experiment – methods (CAPS assay), interpretation of results o HapMap project, trios etc. o Potential effects of mutations on protein sequence What is the definition GMO? Expressing eukaryotic proteins in bacteria (p 190-192) RNAi (p 271-273) o Mechanism of action o Function of all enzymes (Drosha, Dicer, RISC, etc.) o How RNAi experiments are done in C. elegans o Vector used to express dsRNA in bacteria o T7 promoter and T7 polymerase – why and how are these used? (p 195-198) o siRNA versus microRNAs o processing of pri- versus pre-microRNA, etc. o Why is a SNP in a seed sequence so problematic? Gene expression (p 167-173) o Why measure gene expression using mRNA levels? o Explain Northern blot vs. qRT-PCR of measuring mRNAs: pros and cons of different techniques, uses, how they are different/same as each other o General outline of steps/reagents used in Northern blots o qRT-PCR theory, reagents and steps in the reaction (p 123-125) o Understand what the threshold cycle (Ct) is, be able to determine the relative amount of product between samples with different Ct values o Be able to interpret qRT-PCR data and calculate fold-difference between samples o Understand what the t-test tells you Questions on miRNAs and cancer paper by Kefas et al. All calculations used in course! o Conversions and dilutions o Calculate volume of a reagent or amount to add to a digest/ligation given concentration o Molar ratios o Transformation efficiency Note: part of your test will be done on a computer in the computer lab. Make sure you know how to do a BLAST (like in HW2 or in the barcoding bioinformatics exercise), interpret BLAST results, use NEB Cutter, ORF Finder, and ClustalW.

BIMM | 101 | Recombinant DNA Techniques | study guides | o What do the BLAST results te

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