DNA sequencing and application Flashcards
what is Fred Sanger’s DNA sequencing
what is sequencing
-technique that allows genes to be isolated and read
-DNA is cut into fragments and sequencing carried out a number of times on overlapping strands ensures accuracy
-sequencing= working out the nucleotide sequence of a strand of DNA (AGCT)
what are the steps for DNA sequencing
- label 4 test tubes A,T,C,G and into each add:
-sample of DNA to be sequenced
-a radioactive primer= make DNA visual later on gel and enables DNA to be copied
-the 4 nucleotides
-DNA polymerase - add small amount of modified (dideoxy), radiolabelled nucleotide that cannot form phosphodiester bond= no more bases can be added whilst incorporated into synthesis complimentary strand
3.DNA polymerase synthesises many copies of DNA sample= thousands of DNA fragments of different lengths are generated
=in tube A= stop at nucleotide A, Tube T= stop at T etc
4.contents of all tubes are run side by side on electrophoresis gel, DNA bands are visualised by autoradiography
how do you read a chain termination sequencing and gel electrophoresis
either starting at top or bottom of paper= read in order of the blocked squares for each bases= shortest fragments travel the furthest= 1st in chain
describe the differences between thin layer chromatography and electrophoresis
TLC
-separates by relative solubility
-no electricity used
-separates non-charged [articles
-no buffer solution
-dyes used in TLC
-not automated/ computerised
electrophoresis
-separates by size
-electricity used
-only separates charged particles
-buffer solution used
-computerised
what can be used instead of radioactivity used to label the terminal (last added) bases and how does it work
automated sequencing
-fluorescent dyes used instead = glow when scanned with a laser beam and light signature identified by computer
-The polymerisation reaction is done in a single tube, using PCR-like cycles to speed up the process.
-this technique can sequence 12000 bases per minute
explain how the automated sequencing machine orders the DNA fragments from the PCR reaction into the size order shown in fig 3.2
-electrophoresis
-negatively-charged DNA moves towards positive electrode
-smaller fragments move faster
-resolution on gel sufficient to register 1 nucleotide
what is high-throughput sequencing
example is pyrosequencing
This involves sequencing by synthesising a single strand of DNA (complementary to the strand to be sequenced), one base at a time whilst detecting, by light emission which base was added at each step
what is the human genome project and what has this allowed
-completed sequencing the 24000 human genes in human genome in 2003
allowed:
-genome-wide comparisons of individuals and species
-prediction of amino acid sequences in polypeptide
-development of synthetic biology
what is comparative genomics
-comparing genome size. number of genes and chromosome number
-DNA sequence comparison between species
what is an epigenome and how is it affected
-second layer of chemicals that cover DNA and histones, shapes physical structure of genome, tightly wraps inactive genes= unreadable and relaxes active genes
-reacts to external factors e.g diet and stress
what are SNPs
copying errors causing variation in DNA sequence at particular locations
how can primary structure of a protein from organism’s sequenced genome be determined
DNA bases= code for specific codons= codons correspond with an amino acid
what is synthetic biology and how can it be applied
branch of science concerned with designing and building useful biological devices and systems
uses:
-information storage e.g converting classical sources e.g images into specialised compressed codes that can be represented by four alphabet numbers
-production of medicine= biosynthesis of drugs that are too complicated/ expensive to produce or rely on rare plant
-novel proteins
-biosensors= converting environmental info into biochemical info
-nanotechnology
