DNA sequencing and the Human Genome project Flashcards
What is required for Sequencing of DNA?
- DNA fragment of interest amplified by cloning or PCR
- Denatured to serve as template for replication reaction (i.e. single
stranded DNA) - in vitro system for DNA replication / polymerisation
- high through-put and accurate method
DNA Sequencing by the chain termination method
‘Sanger Method
DNA is synthesized in vitro in a mixture that contains ssDNA, DNA polymerase
and the 4 dNTPs
• If a ddNTP analog of one dNTP is present, it can become incorporated into
growing chain
• Because it lacks the 3’OH, the addition of the next dNTP is impossible
• DNA chain terminates at that point
Reaction mixture with ddATP will produce a set
of DNAs of different lengths terminating at
each of the different As
• Exact length of DNA synthesis can be used to
determine the position of each A in the
growing chain
• Repeated for ddCTP, ddGTP and ddTTP
Difference between dideoxyribonucleoside and deoxyribonucleoside
OH at 2’ in RNA which is missing in DNA
In dideoxyribonucleoside triphosphate the OH at 3’ is
missing hence nucleotides cannot be added to the chain
if the OH at 3’ is missing the replication/addition
of nucelotide will stop
Sequencing I
To determine complete sequence, dsDNA
separated into ssDNA and one of the strands is used for sequencing
• 4 different ddNTPs are used in 4 separate DNA
synthesis reactions
• Each reaction produces a set of DNA copies
that terminate at different points in the sequence
Sequencing Il
•The products of these 4 reactions are separated by
electrophoresis in 4 parallel lanes of a polyacrylamide gel
• Newly synthesized strands are detected by a label- usually isotope P32 or such
• In each lane, the bands represent fragments that
have terminated at a given nucleotide but at different
positions in the DNA
•By reading off the bands in order, starting from the bottom of the gel and reading across all 4 lanes, the DNA sequence of the newly synthesized strand can
be determined bottom (5’ – up 3’)
• The sequence is complementary to the template strand
Automation of DNA Sequencing
Modifications to the Sanger method:
• primer is not radioactive • 4 ddNTPs each carry a different coloured fluorescent tag • reaction performed in single tube • fragments separated in single lane • automatic laser scanning and recording
Applications of sequencing in the laboratory
Verify what you have made is what you thought it was
– No base insertions, deletions, substitutions
– Primer bound in right place
– Verify Next Generation Sequencing (NGS)
Shotgun Sequencing
• Longer sequence subdivided into smaller random
fragments
• Sequenced using the chain termination method
• Multiple overlapping reads for the target DNA are
obtained over several rounds of fragmentation and
sequencing
• Computer programs used to piece overlapping ends
together into a continuous sequence
• Enabled full genome sequencing
Does chain termination work for longer strand?
NO
Chain termination method only works for fairly short
strands (100 to 1,000 base pairs)
Sanger sequencing
- sequencing single gene
- sequencing 1-100 amplicon targets at the lowest cost
- sequencing up tp 96 sample at time without barcoding
- microbial identification
- Fragment analysis - high genotyping
- microsatellite or STR analysis
- NGS confirmation
Next generation sequencing
- interrogating >100 genes at a time cost effectively
- finding novel variant by expanding the number of targets sequenced in a single run
- sequencing samples that have low input amounts of starting material
- sequencing microbial genomes for pathogen subtyping to enable research of critical outbreak situation
Where are the genes?
Only one strand serves as a template, but it may be a
different one for different genes
the two strands are not identical they code for different gene
different genes in the ss DNA strand
genes can be overlaping ( in viruses commenly)
Genes in Human Chromosome
the genes are like much less close together
- Large regions of non-coding DNA
- RNA processing (intron/exon)
- Related genes on different chromosomes
- Determining the limits of a gene
How do we determine the function of genes?
1-Relationships within a family of genes: sequence
homology => comparative genomics
2-First member of a gene family: mutation analysis (i.e.
remove gene or change its DNA sequence ® alteration in function at the level of the cell or organism?)
=> genetics / biochemistry
Conservation of Genes
Despite this diversity, critical genes are highly conserved and can be tracked from yeast to human
• e.g. cytochrome c from the electron transport in mitochondria (generation of ATP)
