Biochemistry 2280A Study Guide - Final Guide: Chain Termination, Personalized Medicine, Autoradiograph
30 Nov 2015
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11/30/2015 11:55:00 PM
2280 Topic 27
- Only way to ensure no errors is to sequence it
- Two methods in the early 1970s to sequence DHA
Chemical sequencing (Maxam and Gilbert) → not used often
Dioxy or chain termination sequencing
Chain termination sequencing:
- Involves the enzymatic synthesis of a DNA strand in the presence of base
specific chain terminators
2’3’ Dideoxnucleotides
Deoxy NTPs (ddNTPs) act as chain terminators in synthesis reaction
didioxy → uses terminators
Materials required to sequence a DNA
- DNA to be sequenced (template)
- Oglionucleotide primer that anneals to the template
- DNA polymerase
- dNTPs (G,A,T,C)
- ddNTPs (small amount .2% of dNTP)
Extension
At 1-500 probability the didioxy will go across a T and the chain will stop at
this position
Always goes to a T but don’t know which one
At each base where a dA should be inserted into the growing chain, there is
1/500 chance a ddA will be inserted and the chain terminated
A collection of DNA molecules is made that all have the same 5’ end but
differ at the A position at which they stop (therefore differ at 3’ end)
Do 4 separate reactions, each with a different ddNTP (G,C,T, A)
Done in the presence of radio activity → so they are labeled
Autoradiography of a sequencing experiment
Typically ~300 bases can be read on sequencing gel
Then denature DNA molecules
Then analyze the products by gel electrophoresis
Read the DNA sequence as a ladder from 5'-3’
find more resources at oneclass.com
find more resources at oneclass.com
If you added too little of the ddNTPs to a sequencing reaction, what would
happen?
The chains would never stop and no sequence could be read
The sequence would read as normal
The probability of stopping the chain would increase and a few bases would
be read
- In the mid 1990s automated DNA sequencing became common place. Each
ddNTP is labeled with a different dye that fluoresces at a different
wavelength
Can read about 1000 bases on a good one
Next generation DNA sequencing
Novel methods are now available to obtain large amounts of DNA sequence
at minimal cost
$1000 genome and personalized medicine
RNA profiling (RNA-seq)
pg 343
When expressing your favorite protein in E.coli
- Human and E.coli promoter structures are different
- Introns are found in human genes
- Prokaryote and eukaryotic cells differ in their mechanism of translational
initiation
- Different post translational modifications occur in prokaryotic and
eukaryotic cells
Ex if YFG needs a specific phosphorylation
These two issues are handled through the plasmid used for cloning
- Translation → Prokaryotes have binding sites
Producing YFP
- Purify protein in E. coli
- Check protein for purity and activity
Site directed mutagenesis (custom designing the YFG)
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Only way to ensure no errors is to sequence it. Two methods in the early 1970s to sequence dha. Chemical sequencing (maxam and gilbert) not used often. Involves the enzymatic synthesis of a dna strand in the presence of base specific chain terminators. Deoxy ntps (ddntps) act as chain terminators in synthesis reaction didioxy uses terminators. Oglionucleotide primer that anneals to the template. At 1-500 probability the didioxy will go across a t and the chain will stop at this position. At each base where a da should be inserted into the growing chain, there is. 1/500 chance a dda will be inserted and the chain terminated. A collection of dna molecules is made that all have the same 5" end but differ at the a position at which they stop (therefore differ at 3" end) Do 4 separate reactions, each with a different ddntp (g,c,t, a) Done in the presence of radio activity so they are labeled.
