DNA Sequencing 2009 10

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DNA sequencing

Extended Elective StudiesDNA analysis, Proteomics and MetabolomiASM013 - 2009/10

Dr Giovanna Bermano – [email protected] - Room A35a



DNA Sequencing

• Important to know the precise order of nucleotides in DNA as:

- gives clues about function of gene product responsible fordisease;

- allows comparison of gene sequence with sequences ofgenes in databanks and possible discovery of cause ofdisease;

- allows comparison of a newly discovered gene topreviously discovered genes in other organisms which mayreflect common functions for the protein products.

• Two methods:

- Enzymatic method of Sanger-Coulson (commonly used)

- Chemical degradation method of Maxam and Gilbert



Sanger-Coulson method

• ssDNA used as template.

• Sequencing primer anneals to ssDNA. Primer is complementary tothe 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 specificddNTPs (dideoxynucleotide) to terminate enzymatically thereaction.

DNA Sequencing



• The reactions are run inthe presence of ddNTPs.

• This is just like regularnucleotides, except it has

no 3' hydroxyl group -once it is added to the endof a DNA strand, it can nolonger be elongated.

DNA Sequencing



The reaction mix includes

• the template DNA,

• free nucleotides,

• an enzyme (usually a variantof Taq polymerase)

• a 'primer' - a small piece of

single-stranded DNA about20-30 nt long that canhybridize to one strand of thetemplate DNA.

DNA Sequencing



DNA Sequencing



• Gel electrophoresis canbe used to separate thefragments by size.

• This diagram shows theresults of a sequencing

reaction run in thepresence of dideoxy-Cytidine (ddCTP).

• This can be repeated forall four ddNTPs.

DNA Sequencing



• All four reactions can be runin a single tube with “all four”of the ddNTPs (A, G, C andT) present, and with“different” fluorescentcolours on each.

• The sequence of the DNA isdetermined by knowing thecolour codes.

• The gel is read from bottomto top: TGCGTCCA-(etc).

DNA Sequencing



• The computer reads the sequence from the gel byscanning, from smallest fragment to largest, the coloursin one lane of a gel (one sample).

• The computer also produces a text file containing thenucleotide sequence.

• An average of 500 nucleotides are read in eachreaction.

• DNA sequencinghttp://uk.youtube.com/watch?v=Mz-4LSfecM4&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 bychemical degradation, followed by high resolution acrylamide gelelectrophoresis.

• This method involves base specific cleavages:

- base first modified using specific chemicals- the sugar phosphate backbone of the DNA is then cleaved bypiperidine 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

DNA Sequencing



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.



Deriving meaningful knowledge from DNA sequences will require theexpertise and creativity of teams of biologists, chemists, engineers, andcomputational 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 post-

translational events

– Interaction of proteins in complex molecular machines

Research challenges in 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, including microbial consortia useful

for environmental restoration – Developmental genetics, genomics



High quality draftMouse genome2002

Partially sequencedDog genome2003

2.8 x 109Rat genome2004

3 billionHuman genome2006

3.3 x 109Human genome2001

4600000E.coli 1997

12500000S. cerevisie 1996

49000Bacteriophage λ1982

16600Human mitochondria1981

4363pBR3221978

5243SV401977

5386Bacteriophage ФX1741975

Size (bp)OrganismYear

Genome Analysis



What Does the Human Genome Sequence Tell Us?

By the Numbers

• The human genome contains 3164.7 million chemical nucleotidebases (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 lowerthan previous estimates of 80,000 to 140,000.

• Almost all (99.9%) nucleotide bases are exactly the same in allpeople.

• 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 ("junkDNA") 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 tohave occurred in the rate of accumulation of repeats in thehuman genome.



How the Human Compares with Other Organisms

• Humans have on average three times as many kinds of proteins as thefly or worm because of mRNA transcript "alternative splicing" andchemical modifications to the proteins.

• Humans share most of the same protein families with worms, flies, andplants, but the number of gene family members has expanded inhumans.



Variations and Mutations

• Scientists have identified about 1.4 million locations wheresingle-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 inmales vs females. Researchers point to several reasons for thehigher mutation rate in the male germline, including the greaternumber of cell divisions required for sperm formation than for

eggs.



Applications, Future Challenges

• The genome sequence will help on finding genes associatedwith disease.

• A number of genes have been associated with breast cancer,muscle disease, deafness, and blindness.

• Finding the DNA sequences underlying such commondiseases as cardiovascular disease, diabetes, arthritis, andcancers will provide focused targets for the development ofeffective new therapies.

• It will be possible to study all the genes in a genome, forexample, 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 workwith each other and the environment to create complex, dynamic livingsystems.

• These explorations will encompass studies in transcriptomics,

proteomics, structural genomics, new experimental methodologies, andcomparative genomics.

• Transcriptomics involves large-scale analysis of messenger RNAstranscribed from active genes to follow when, where, and under whatconditions genes are expressed.



• Proteomics involves the study of protein expression and

function.

• Structural genomics involves the generation of the 3-Dstructures of proteins, offering clues to function and biologicaltargets for drug design.

• Knock out studies will be used to understand the function ofDNA sequences and the proteins they encode.

• Comparative genomics involves the analysis of DNAsequence patterns of humans and well-studied modelorganisms for identifying human genes and interpreting their

function.



Human Genome Project Information

http://www.ornl.gov/sci/techresources/Human_Genome/faq

/seqfacts.shtml#whatis

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DNA Sequencing 2009 10