BIOLOGY 305 1st Edition Lecture 16 Outline of Last Lecture I. Introduction to Genetic InformationII. DNA as the Source of Genetic InformationA. Nucleotide Building BlocksB. Helical StructureIII. SupercoilingIV. More about Genetic MaterialOutline of Current Lecture I. Introduction to Methods Studying GenesII. Polymerase Chain ReactionIII. Analysis of DNA, RNA, and ProteinIV. Restriction AnalysisV. High Throughout SequencingVI. Vocabulary and Sample QuestionsCurrent LectureI. Introduction to Methods Studying GenesMechanism is more important than methods, but the exams will use methods to test your knowledge of mechanism. Reading the whole chapter 10 is required for this lecture.It is difficult to study genes because they are so small. There is no way to look at a single molecule and see its mechanism and sequence and etc. The most realistic way for gene analysis is to analyze large pools of molecules.How do we study genes and gene products?1) Have to be able to section and single about specific fragments2) Have to be able to read RNA involved3) Have to be able to analyze productsWhat is the basic approach to obtain a DNA sequence?1) First, generate random fragments of DNA- Isolating the DNA by digestion of restriction enzymes2) Cloning - Generate a large sample of identical sequences (ligated into plasmids and amplifiedin bacteria3) Identification - By colony hybridization, complementation, DNA sequencingThe most modern way to obtain a DNA sequence is to search a genome database (such as NCBI)these resources are useful for PCR and DNA SynthesisHow is DNA introduced into organisms?1) Take obtained DNA2) Add additional sequences- Usually via plasmid, which helps to transfer and integrate DNA into the host genome3) Transfer into organism- Shock the cells to increase permeability for DNA uptake (Ex: electroporation)Three important approaches for DNA analysis:- PCR and Sequencing, Southern Blot, High Throughput sequencingII. Polymerase Chain ReactionPolymerase Chain Reaction (PCR) – sequence a small region with defined endsPurpose: use specially designed primers for direct amplification of specific short regions of DNA in vitroApplications: detect mutations, recombine DNA, for identification in crime investigation, - Doesn’t rely on bacteria, cells, or restriction like the basic method doesmeans you can do this (amplify DNA) entirely in-vitro!- Generate a large amount of DNA using at least 2 types of primers- Primers determine specificity of DNA to be amplified: if sequence is close together are only present once in the whole genome, the only DNA segment able to amplified is the one between the two primersWhat are the typical key reagents of PCR?- Genomic DNA templatecontains region that we want to be amplified- PrimersCalled ‘sense’ primer if the sequence is identical to the mRNA copy translated into a protein, ‘anti-sense’ refers to the sequence on the opposite strand- dNTPsNecessary nucleotide building blocks- Mg2+DNA cofactor, Stabilizes binding of enzyme to template DNA- Taq PolymeraseIsolated from an organism that lives in extreme temperature. It’s optimum temperature is around 75 degrees CelsiusMethod:1) Some PCR protocol requires an initial denaturationCycle 1:A) Start with a double-stranded DNA template and denature it with heat (usually 95 C), Separates the strands as heat disrupts the hydrogen bonds between complementary bases. Then, anneal the primers with colder temperature (usually) 55-60 CB) Extend the primers (optimal temperature for DNA polymerase is 70C)Often using Taq PolymeraseCycle 2:C & D) Denature, anneal, and extendThe first and second strands get amplified (4x by the end of this cycle)Cycle 3: E-G) After repetition, you will have the exact desired length of DNA:Reasoning: Pieces that are too long, amplification is linear, not exponentialBut short pieces ARE exponentially amplified, thus accumulate much more through the chain reaction and can be detectedTypically do this for 15-35 cyclesEstimating number of copies PCR per cycle: (# initial DNA molecules) * 2nWhere n is number of cyclesRT-PCR – reverse transcription polymerase chain reactionPurpose: Amplify starting with RNAThe difference is that there are a few steps before the PCR reaction: RNA must be turned into DNA. Using reverse transcription, a cDNA copy can be made from RNA and the cDNA can be used in a subsequent PCRIII. Analysis of DNA, RNA, and ProteinWhat are the different approaches to quantify classes of genes and gene products?DNA – Southern blot, High-throughput sequencingRNA – Northern blot, RT-PCR, HTSProtein – Western blotBlotting:Southern blot – to find length of restriction fragments1) Each starts with genomic DNA from an organism, which is digested with restriction enzymes that recognize specific short sequences2) The DNA is separated on a gel. Separating on a gel separates the sequences by size by applying a electricalgradient that pulls the negatively charged molecules through the medium at different rates according to the molecule’s molecular weight3) Transfer of DNA to a membrane (aka blotting it to a piece of paper)4) Hybridization – DNA is double-stranded so we can anneal the sample DNA to a radioactively attached probeThe regions that light up indicate complementationNorthern Blot – similar method, except using RNADifference: when there is matching RNA, you will still see a signal.Ex: if it migrates to about between 4 and 5 kb, we can conclude its size is 4.5Note: You can measure gene expression by looking at level of RNAWestern blot – similar to southern blotMain difference: we use antibodies as the probe (radioactive probes are for DNA) Dideoxy/Sanger sequencing – determines single nucleotide sequence of a DNA segmentWhat are the required reagents? Primer, DNA Polymerase, template DNA, all four dNTPs, all four(fluorescently labeled) ddNTPs in separate tubesIV. Restriction AnalysisYou digest a plasmid with Xba I. In a separatereaction, you digest the same plasmid with Spe I.Finally, you perform a double digest. You run yourdigests on a gel. Draw your results.a) A labeled primer is used to initiate DNA synthesis and the addition of dideoxynucleotides (ddATP) randomly arrests synthesis.Basically the ddATP ‘snips’ the DNA into piecesb) The fragments are separated using electrophoresis and are subjected to autoradiography.The inferred sequence is the OPPOSITE from original strand:To read a Sanger sequences, starting at the top of the gel to