Technologies of DNA sequencing Exam Bank Solution Manual Already Passed

Technologies of DNA sequencing Exam Bank Solution Manual Already Passed DNA structure - Answers Nucleotides - purine, pyramidine DNA is always written 5' to 3' 5' relates to the phosphate to 3' hydroxy - all nucleotides are written in this way Polymerase always adds the next nucleotide to the 3' end. So the DNA extends 5' to 3' with the polymerase Sanger sequencing - Answers Still used a lot today despite the new generations fo sequencing Illuminati sequencing - Answers Predominant form of next generation sequencing which is predominantly used for diagnostics around the world now DNA can because - Answers can be denatured to single strands via heat /chemical • bond between 5' phosphate and 3' hydroxy is crucial • utilized in Sanger sequencing and in engineering (DNA ligation) Bonds between the bases - Answers G and C have 3 bonds AT - 2 bonds If seperate the strands GC and are bound together with a higher affinity than AT - which matters for PCR - denature DNA by heating it up and treating with alkylate and it falls apart and you end up with single stranded DNA Have the 3' hydroxy and then you added the 5' phosphate fo the next nucleotide you are adding at it extends on like a zip Role of DNA polymerase in sequencing - Answers DNA polymerase 2 and 3 -In order for DNA polymerase to work needs to make a double stranded DNA called a primer - forms by annealing the phosphate bonds across the two strands and then that primes, will extend in the right temp and conditions - example is PCR. XAdding nucleotides to other strand with DNA polymerase 3 - catalyses formation of the phsophodiestwer bond ( phosphate hydroxy bond) extending DNA . DNA polmymerase 1 and 3 has proof reading - high accuracy. DNA - template non- template - Answers Have a template strand and are adding the complement of it with DNA polymerase 1 or DNA polymerase 3 and it catalyses the formation of the phosphodiester bond which is the phosphorescent hydroxy bond and extends the DNA. DNA polymerase 1 and 3 had proof reading so if the wrong nucleotide base is added it can remove it and add the next nucleotide. In order for DNA polymerase to work you must have a double stranded piece fo DNA as it cannot just make it up, so it has to have a starting point which is called a primer if we are adding it. Primer - Answers Piece of single stranded DNA that will form by annealing the phosphate bonds across the two strands and then that primes. If you have nucleotides and primers in the solution and set at the right temperature and the right condition it will extend What does DNA polymerase do - Answers Catalyses the formation of the phosphodiester bond are accurate and can also proof-read can be used in engineering/sequencing applications requires a primer (oligonucleotide) What lots of a specific sequence - Answers Amplify via PCR Use a heat stable polymerase such as Taq (so can work at high temperatures) Short pieces of DNA can be synthesized chemically (eg. use as prim To sequnce something - Answers Typically we make a copy (using DNApol) and sequence the copy PCR - Answers Can make lots of DNA Process is denature annealing extension 1. Add oligonucleotide primers - these are single stranded DNA primers which already know the sequence of 2. Heat to seperate strands (95deg) 3. Cool to that primerscan anneal (55-65 deg) 4. Heat to 72 deg to allow DNA synthesis Repeats steps 2 and 3 Allows extension lots of copies After 25 cycles the target sequence has been amplified about 10^6 fold. Polymerase come along and inceorpperate the new nucleotides i nthe solution Usually do about 30 cycles Relavant for sequencing PCR process - Answers Have two primers because we already know the sequence somehow so can make the single stranded DNSA primers. We danature the DNA and we allow the primers to anneal. In the right condition with nucleotides and magnesium and the polymerase you make a copy of the target DNA you want to amplify. And then you cycle the process, and eventually the PCR is just amplify ing product you have already made and not the original template. Three steps of CPR - Answers Denaturing - making the DNA single stranded Annealing - primers anneals Extension - polymerase comes along and incorporates the new nucleotides dint odour solution Normally do about 30 cycles Bacteria / prokaryotes - Answers E,coli 4.7 Mb (4.7 x 10^6) = genome size 4,288 genes Plasmid - Answers Can replicate inside Ecoli and is usually about BP so about 1000 x smaller roughly than an ecology genome Eukaryotes - Answers Humans. Genome size = 3.2Gb = 3.2x10^9 BP Genes = 21,000 1000 x more complex than an Ecolis genome Sanger sequencing - Answers Dideoxy chain termination Incorporation of dideoxy nucleotide protein leads to termination of nascent chain. In sequencing reaction have a small number of dideoxy nucleotides - mostly deoxynucleotides so can be extended But if put a fluorescent die on the dideoxy nucleotides - see different colour dyes for each of the dideoxy nucleotide Polymerase incorporates the dideoxy nucleotides - only happens very few times - in every extension end up with a small number which you have extended which you stop where all others have extend - end up with lots of different size products which stop when you have incorpreated the dideoxy nucleotides - end up with a whole pool of sequences Sanger sequencing process - Answers Still used today - same concepts used for illumination sequencing made nucleotides which only have a H instead of a hydroxy called a dideoxynucleotide meaning it cannot be extended as the next phosphate cannot be bound. In the sequencing reaction have a small number of dideoxynucleotides - mostly they are not (mostly they are deoxynucleotide which have the OH hydroxy and so can't be extended). Put a fluorescent dye on the didepoxynucleotdies and use four different dyes for each fo the nucleotides. The polymerase only incorporates the dideoxynucleotides a small amount of times as only put a small amount of tin the tube. End up with lots of different sized extended products due to the incorporation of the dideoxynucleotdies, load this DNa into a capillary or a gel and run it down, small stuff fast and large slower -separated by size , shine a layer on it and the layer reads the colour. So can read the sequence based on the colour