DNA sequencing

Extended Elective Studies DNA analysis, Proteomics and Metabolomics ASM013 - 2009/10 Dr Giovanna Bermano – g.bermano@rgu.ac.uk - Room A35a

DNA Sequencing
• Important to know the precise order of nucleotides in DNA as: - gives clues about function of gene product responsible for disease; - allows comparison of gene sequence with sequences of genes in databanks and possible discovery of cause of disease; - allows comparison of a newly discovered gene to previously discovered genes in other organisms which may reflect common functions for the protein products. • Two methods: - Enzymatic method of Sanger-Coulson (commonly used) - Chemical degradation method of Maxam and Gilbert

each reaction uses a specific ddNTPs (dideoxynucleotide) to terminate enzymatically the reaction. • Based on premature termination of DNA synthesis. Primer is complementary to the region near the vector-insert junction. • Taq or T7 DNA polymerase extend the primer using dNTPs. • Extension reaction is split into 4. • Sequencing primer anneals to ssDNA. .DNA Sequencing Sanger-Coulson method • ssDNA used as template.

except it has no 3' hydroxyl group once it is added to the end of a DNA strand.DNA Sequencing • The reactions are run in the presence of ddNTPs. • This is just like regular nucleotides. . it can no longer be elongated.

.DNA Sequencing The reaction mix includes • the template DNA.a small piece of single-stranded DNA about 20-30 nt long that can hybridize to one strand of the template DNA. • free nucleotides. • an enzyme (usually a variant of Taq polymerase) • a 'primer' .

DNA Sequencing .

. • This can be repeated for all four ddNTPs.DNA Sequencing • Gel electrophoresis can be used to separate the fragments by size. • This diagram shows the results of a sequencing reaction run in the presence of dideoxyCytidine (ddCTP).

DNA Sequencing • All four reactions can be run in a single tube with “all four” of the ddNTPs (A. C and T) present. and with “different” fluorescent colours on each. . • The gel is read from bottom to top: TGCGTCCA-(etc). G. • The sequence of the DNA is determined by knowing the colour codes.

• The computer also produces a text file containing the nucleotide sequence. • DNA sequencing http://uk.youtube.DNA Sequencing • The computer reads the sequence from the gel by scanning.com/watch?v=Mz4LSfecM4&feature=PlayList&p=BADA17575EBD7A76&playnext=1&index=8 . • An average of 500 nucleotides are read in each reaction. from smallest fragment to largest. the colours in one lane of a gel (one sample).

DNA Sequencing .

the sugar phosphate backbone of the DNA is then cleaved by piperidine at that site.DNA Sequencing Maxam and Gilbert method • Based on introduction of strand breaks at specific nucleotides by chemical degradation.base first modified using specific chemicals . • Used for sequencing of particular genes whose sequence is GC rich. . • This method involves base specific cleavages: . followed by high resolution acrylamide gel electrophoresis. • Not commonly used as it is slower than Sanger method.

DNA Sequencing Maxam and Gilbert method .

.protein function and cellular pathways.the evolution of gene/protein families.pathogen/host relationships. . .Genome Analysis • To know the sequence of the entire genome of an organism is useful for several reasons and it provides a better understanding of: .gene interactions and the regulation of gene expression. . and hence. organisms.

chemists. and functions Gene regulation DNA sequence organization Chromosomal structure and organization Noncoding DNA types. among others. exact locations. protein synthesis. engineers. amount. and posttranslational events – Interaction of proteins in complex molecular machines . and functions – Coordination of gene expression.Research challenges in genetics Deriving meaningful knowledge from DNA sequences will require the expertise and creativity of teams of biologists. and computational scientists. distribution. information content. What we still do not know: – – – – – Gene number.

genomics . including microbial consortia useful for environmental restoration Developmental genetics.– – – – – – – – – Predicted vs experimentally determined gene function Evolutionary conservation among organisms Protein conservation (structure and function) Proteomes (total protein content and function) in organisms Correlation of SNPs (single-base DNA variations among individuals) with health and disease Disease-susceptibility prediction based on gene sequence variation Genes involved in complex traits and multigene diseases Complex systems biology.

cerevisie E.Genome Analysis Year 1975 1977 1978 1981 1982 1996 1997 2001 2002 2003 2004 2006 Organism Bacteriophage ФX174 SV40 pBR322 Human mitochondria Bacteriophage λ S.3 x 109 High quality draft Partially sequenced 2.coli Human genome Mouse genome Dog genome Rat genome Human genome Size (bp) 5386 5243 4363 16600 49000 12500000 4600000 3.8 x 109 3 billion .

9%) nucleotide bases are exactly the same in all people.000. • The functions are unknown for over 50% of discovered genes. T.7 million chemical nucleotide bases (A. but sizes vary greatly • The total number of genes is estimated at 30. . • Almost all (99. C. much lower than previous estimates of 80.000 to 140.000. • The average gene consists of 3000 bases.What Does the Human Genome Sequence Tell Us? By the Numbers • The human genome contains 3164. and G).

but they shed light on chromosome structure and dynamics. • Repetitive sequences are thought to have no direct functions.• Less than 2% of the genome codes for proteins. • Repeated sequences that do not code for proteins ("junk DNA") make up at least 50% of the human genome. . a dramatic decrease seems to have occurred in the rate of accumulation of repeats in the human genome. • During the past 50 million years.

• Humans share most of the same protein families with worms. and plants.How the Human Compares with Other Organisms • Humans have on average three times as many kinds of proteins as the fly or worm because of mRNA transcript "alternative splicing" and chemical modifications to the proteins. . but the number of gene family members has expanded in humans. flies.

4 million locations where single-base DNA differences (SNPs) occur in humans. Researchers point to several reasons for the higher mutation rate in the male germline. including the greater number of cell divisions required for sperm formation than for eggs.Variations and Mutations • Scientists have identified about 1. . • The ratio of germline (sperm or egg cell) mutations is 2:1 in males vs females. • This information promises to revolutionize the processes of finding disease-associated sequences.

• It will be possible to study all the genes in a genome. • Finding the DNA sequences underlying such common diseases as cardiovascular disease. Future Challenges • The genome sequence will help on finding genes associated with disease. muscle disease. and blindness. for example. arthritis. • A number of genes have been associated with breast cancer. and cancers will provide focused targets for the development of effective new therapies. . diabetes. or all the transcripts in a particular tissue or organ. deafness.Applications.

and under what conditions genes are expressed. structural genomics. proteomics. . new experimental methodologies. and comparative genomics. • Transcriptomics involves large-scale analysis of messenger RNAs transcribed from active genes to follow when. where. dynamic living systems. • These explorations will encompass studies in transcriptomics.The Next Step: Functional Genomics • To use this vast reservoir of data to explore how DNA and proteins work with each other and the environment to create complex.

• Comparative genomics involves the analysis of DNA sequence patterns of humans and well-studied model organisms for identifying human genes and interpreting their function. • Knock out studies will be used to understand the function of DNA sequences and the proteins they encode. • Structural genomics involves the generation of the 3-D structures of proteins. offering clues to function and biological targets for drug design. .• Proteomics involves the study of protein expression and function.

ornl.Human Genome Project Information http://www.shtml#whatis .gov/sci/techresources/Human_Genome/faq /seqfacts.

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