* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download DNA sequencing File
DNA barcoding wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Maurice Wilkins wikipedia , lookup
Whole genome sequencing wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Agarose gel electrophoresis wikipedia , lookup
DNA sequencing wikipedia , lookup
Genome evolution wikipedia , lookup
Transformation (genetics) wikipedia , lookup
SNP genotyping wikipedia , lookup
Real-time polymerase chain reaction wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Molecular cloning wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
DNA supercoil wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Molecular evolution wikipedia , lookup
Non-coding DNA wikipedia , lookup
Genomic library wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Community fingerprinting wikipedia , lookup
DNA sequencing Genome Sequencing • The genome is all of the genetic information within an organism. • The term can variously be used to mean: – All of the genes in the chromosome of an organism, or – The genes carried in the nucleic acid of a virus and the DNA of mitochondria and chloroplasts • Viruses, mitochondria and chloroplasts do not have chromosomes, but do have nucleic acids which carry genes. • The Human Genome Project (HGP) has successfully identified, mapped and sequenced the entire human genome. – Makes it possible to use this information for various purposes, e.g. • Research into gene regulation and functioning • Construct gene probes for identifying particular genes and abnormalities Gene Sequencing • Automated gene sequencing uses a combination of interrupted PCR or chain termination and electrophoresis. • DNA to be sequenced is denatured and mixed with: • Primer (of around 20 nucleotides) • DNA polymerase, • DNA nucleotides (deoxyribose nucleoside phosphates or dNTPs) • Modified DNA nucleotides (dideoxyribose nucleoside phosphates or ddNTPs) • There are four dNTPs – dATP, dGTP, dTTP and dCTP • And there are four corresponding ddNTPs: – ddATP, ddGTP, ddTTP and ddCTP –each of which is tagged with a different fluorescent dye • The primer attaches to its complementary sequence which enables replication. • DNA nucleotides hydrogen bond with their complementary bases in the single-stranded DNA. • DNA polymerase puts a covalent phosphodiester bond into the sugar phosphate backbone in the usual way. • Sometimes a modified nucleotide is incorporated: – Stops the replication – Further nucleotides cannot be added as the ddNTPs lack a hydroxyl group so they are unable to bond with another nucleotide. • The process is repeated many times. – Chance determines whether dNTP or ddNTP is incorporated. • Consequently many DNA fragments are produced of differing length. • These fragments can be separated by electrophoresis – Fragments pass through a detector in size order – The colour of the last base inserted is determined – Remember, each of the four ddNTPs has a different colour fluorescent dye attached 1. DNA section is denatured and primed: T A A T C C G G A T T A G C T A A T ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? T A A T C C G G A T T A G C T A A T ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? The DNA is denatured and mixed with primer, dNTPs and ddNTPs, each with a different coloured dye attached. 2. Polymerisation and chain termination: T A A T C C G G A T T A G C T A A T ? C ? T ? T ? A ? ? ? ? ? ? ? ? ? ? ? T A A T C C G G A T T A G C T A ? C ? T ? T ? A ? C ? T ? ? ? ? ? ? ? ? ? T A A T C C G G A T T A G C T A A T ? ? C T ? ? ? ? ? ? ? ? ? ? ? ? ? T A A T C C G G A T T A G C T A ? A T C ? ? ? ? ? ? ? ? ? ? ? ? ? ? T A A T C C G G A T T A G C T A A T ? C ? T ? T ? ? ? ? ? ? ? ? ? ? ? ? T A A T C C G G A T T A G C T A A T ? C ? T ? T ? ? A C ? ? ? ? ? ? ? ? ? ? T A A T C C G G A T T A G C T A A T ? C ? T ? T ? A ? ? T C ? ? ? ? ? ? ? ? A T ? C Chance determines whether a dNTP or a ddNTP nitrogen bonds with its complementary base. When a dNTP bonds, the DNA polymerase can put a covalent bond into the DNA backbone and copying continues. When a ddNTP bonds, the DNA polymerase is unable to put a covalent bond in the DNA backbone and the copied fragment is thrown off. A T G C Dideoxyribose nucleoside phosphates or ddNTPs, each with a different coloured dye attached 3. Electrophoresis and detection of terminal nucleoside: Laser source 7 6 5 4 3 2 1 Detector The DNA fragments are separated by electrophoresis and pass in size order through a colour detector. This determines the colour of the terminal nucleoside. 4. Sequence of colours is the same as the sequence of bases: 1 2 3 4 5 6 7 C T T A C T C If the colours are detected as blue, green, green, …, this corresponds to base order cytosine, thymine, thymine… A T G C Dideoxyribose nucleoside phosphates or ddNTPs, each with a different coloured dye attached • Even with current technology, it is not possible to sequence bases in a long length of DNA, let alone a large gene or an entire chromosome. • Instead, the DNA to be sequenced is cut into smaller fragments and these are sequenced. • A computer program then puts them in order by comparing overlapping sections of code. • Once the order of bases in DNA is sequenced, comparisons can be made within and between species. Such comparisons show: • Some DNA sequences have a lot of variation • Some DNA sequences have very little variation – e.g. the homeobox genes are highly conserved • Similarities, and indeed differences in base sequence, can be used to: – Confirm evolutionary affinities and classification – Suggest how long ago they diverged • e.g. possible evolution of primates • Animations of DNA sequencing: http://www.dnalc.org/resources/animations/cycseq.html http://www.pbs.org/wgbh/nova/genome/media/sequence.swf