Technologies of DNA sequencing Exam Bank Solution Manual Already Passed
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Technologies of DNA sequencing
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Technologies Of DNA Sequencing
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 t...
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
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