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

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

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

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

DNA Sequencing

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). This can be repeated for all four ddNTPs.

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

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

DNA Sequencing

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

DNA Sequencing
Maxam and Gilbert method

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; - protein function and cellular pathways; - the evolution of gene/protein families, and hence, organisms; - pathogen/host relationships.

Research challenges in genetics

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

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, including microbial consortia useful for environmental restoration Developmental genetics, genomics

Genome Analysis
Year 1975 1977 1978 1981 1982 1996 1997 2001 2002 2003 2004 2006 Organism Bacteriophage X174 SV40 pBR322 Human mitochondria Bacteriophage S. cerevisie E.coli Human genome Mouse genome Dog genome Rat genome Human genome Size (bp) 5386 5243 4363 16600 49000 12500000 4600000 3.3 x 109 High quality draft Partially sequenced 2.8 x 109 3 billion

What Does the Human Genome Sequence Tell Us?

By the Numbers The human genome contains 3164.7 million chemical nucleotide bases (A, C, T, and G). The average gene consists of 3000 bases, but sizes vary greatly The total number of genes is estimated at 30,000, much lower than previous estimates of 80,000 to 140,000. Almost all (99.9%) nucleotide bases are exactly the same in all people. The functions are unknown for over 50% of discovered genes.

 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. Repetitive sequences are thought to have no direct functions, but they shed light on chromosome structure and dynamics. During the past 50 million years, a dramatic decrease seems to have occurred in the rate of accumulation of repeats in the human genome.

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. Humans share most of the same protein families with worms, flies, and plants, but the number of gene family members has expanded in humans.

Variations and Mutations

Scientists have identified about 1.4 million locations where single-base DNA differences (SNPs) occur in humans. This information promises to revolutionize the processes of finding disease-associated sequences. The ratio of germline (sperm or egg cell) mutations is 2:1 in males vs females. 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.

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

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, dynamic living systems. These explorations will encompass studies in transcriptomics, proteomics, structural genomics, new experimental methodologies, and comparative genomics. Transcriptomics involves large-scale analysis of messenger RNAs transcribed from active genes to follow when, where, and under what conditions genes are expressed.

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

Human Genome Project Information

http://www.ornl.gov/sci/techresources/Human_Genome/faq /seqfacts.shtml#whatis