DNA Sequencing and the Human Genome Project Flashcards
Biotechnology
Technology:
the application of scientific
knowledge to the practical
aims of human life
Biotechnology,
the use of biology to solve
problems and make
useful products
DNA polymerase
Enzyme (protein) that synthesizes
DNA by joining nucleotides
together using a DNA template as
a guide
ddNTP
- DdNTP refers to Dideoxynucleotides
triphosphates which are used in Sanger
dideoxy method to produce different lengths of DNA strands for DNA sequencing. - DdNTP includes ddATP, ddTTP, ddCTP and
ddGTP. - DdNTP are useful in the analysis of DNA’s structure as it stops the polymerisation of a DNA strand during a DNA replication, producing different lengths of DNA strands
replicated from a template strand
1977 – Sanger Sequencing
The Sanger sequencing method in 7 steps.
(1) The dsDNA fragment is denatured into two ssDNA
fragments.
(2) A fragment of ssDNA is multiplied into millions of copies.
(3) A primer that corresponds to one end of the fragment is attached.
(4) The fragments are added to four polymerase
solutions. Each solution contains the four types of bases but only one type of termination nucleotide.
(5) The chain grows until a termination nucleotide is randomly added.
(6) The resulting dsDNA fragments are denatured to obtain a series of ssDNA of various lengths.
(7) The fragments are separated by electrophoresis and the sequence is read
Sanger DNA Sequencing by
Capillary Electrophoresis
The four solutions of the Sanger method migrate in the same capillary and the four different
fluorescent dye-terminations are detected as they go through the detector.
Sequencing by Synthesis
A) Library Preparation- NGG library is prepared by fragmenting a gDNA sample and ligating specialised adapters to both fragment ends
prepared
B) Cluster Amplification- Library is loaded into a flow cell and the fragments are hybridised into the flow cell surface. Each bound fragment is amplified into a clonal cluster via bridge amplification
C) Sequencing reagents, including fluorescently labelled nucleotides are added and the first base is incorporated. The flow cell is imaged and the emission from each cluster is recorded. The emisiion wavelength and intensity are used to identify the base. This cycle is repeated n times to create a length of n bases
Nnanopore
Nanopore DNA sequencing is a laboratory technique for
determining the exact sequence of nucleotides, or bases, in a
DNA molecule.
Nanopore DNA sequencing involves reading the code of single
DNA strands as they are threaded through extremely tiny pores
(nanopores) embedded within a membrane.
As the DNA moves through the pore, it creates electrical signals
that can be converted to read each base.
This approach offers a low-cost, rapid process for studying long
stretches of DNA.
1976- Bacteriophage MS2
-Bacteriophage containing a single strand of DNA
-1st RNA based genome to be completely sequenced
-Walter Fiers and his team at the University of Ghent, Belgium
-3569 Bases and 1 Chromosome
1977 – The genome Sequenced
By the SANGER Method
PhiX174
-Bacteriophage containing a single circle of DNA
-1st DNA-based genome to be sequences
- Fred Sanger and his team in Cambridge
-5386 Bases and 1 Circular Chromosome
1995 – The first free-living organism
Sequenced
Haemophilus influenza
-non moving rod shaped bacterium that causes meningitis
-1st bacteria to be sequences
-12.1 Million Bases
-32 Chromosomes
1996 – First Eukaryote to be
Sequenced
1998 – First multicellular
Organism Sequenced
2000 – The First Plant and the First
Insect Sequence
2001 – Draft of the Human Genome
was assembled
Clone-by-Clone Sequencing: Mapping
Genome mapping provided the basis for whole genome sequencing and the Human Genome Project
Genetic mapping looks at how
genetic information is shuffled
between chromosomes or
between different regions in
the same chromosome during
meiosis. A process called
recombination or ‘crossing
over’
Physical mapping looks at the
physical distance between
known DNA sequences
(including genes) by working
out the number of base pairs?
(A-T, C-G) between them
Clone-by-Clone Sequencing: Mapping
The top line shows the location of ‘restriction’ sites (vertical
bars) in a particular region of the genome.
The fragments produced by cleavage at every possible point
in this region are numbered 1 to 9.
Below the line are several clones with random end points,
labelled A to F.
Clones are produced by first partially digesting many copies
of the genome with different restriction endonucleases;
The resulting large segments are then inserted into bacteria
and replicated (cloned).
Each clone is digested with a restriction endonuclease, and
the resulting fragments are separated, by size, on an
electrophoretic gel (‘gel analysis of inserts’).
This process yields a distinctive pattern (‘fingerprint’) for
each clone.
The map-assembly problem requires working backwards
(upwards in this figure) from the fingerprints to a clone-
overlap map and
restriction-site map of the chromosome segment.
To finish the analysis of this region of the genome, the
natural choice of clones to sequence would be A and B.
Bacterial Artificial Chromosome (BAC)
A bacterial artificial chromosome (BAC) is an
engineered DNA molecule used to clone DNA
sequences in bacterial cells (for example, E. coli).
BACs are often used in connection with DNA
sequencing.
Segments of an organism’s DNA, ranging from 100,000
to about 300,000 base pairs, can be inserted into BACs.
(11,000 - 32,000 BACs for the entire Human Genome.)
The BACs, with their inserted DNA, are then taken up
by bacterial cells.
As the bacterial cells grow and divide, they amplify the
BAC DNA, which can then be isolated and used in
sequencing DNA.
Clone-by-Clone Sequencing:
Hierarchical Shotgun Sequencing
1- Break genome into large fragments and insert into clones
2- Order clones
3-Break individual clones into small pieces
4- Generate 1000s of sequence reada and assemble sequence of clones
5-Assemble sequences of overlapping cloners to establish reference sequence
1998 - The Advent of CELERA
Whole Genome Shotgun Sequencing.
Rather than the traditional method of separating the
genome into BACs and sequencing each individual BAC via BAC-end Shotgun Sequencing, Myers
proposed the idea of breaking many copies of the entire genome into different sized pieces and
sequencing those pieces in a special way
The principle behind his method was the breaking up
of many copies of the whole genome randomly into
pieces, and then parsing them together, using
repeating elements such as LINEs as identifying markers.
Initially CELERA competed with the Human
Genome Project and planned to pay for the costs
by patenting the whole of the Human Genes
