Download Notions of Biochemistry and Molecular Biology Manipulating DNA

Special course in Computer Science:
Molecular Computing
Lecture 2-3: Notions of Biochemistry and
Molecular Biology, manipulating DNA
Vladimir Rogojin
Department of IT, Åbo Akademi
http://combio.abo.fi/teaching/special
Fall 2013
06/11/2013
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DNA
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DNA
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Base element of Molecular Computing
Importance of DNA
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Responsible for inheritance: encodes all
genetic information of an organism
Responsible for life processes: encodes all
“instructions” needed for the functioning of
the organism
The most fundamental mechanisms of DNA
manipulation are the same in all living
organisms
http://combio.abo.fi/teaching/special
wikipedia
Today we will learn
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Biology of organic
compounds and their
functioning:
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Structure of DNA, RNA,
proteins
Central dogma: how
instructions in DNA are
“executed”
Genes and chromosomes
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Sequencing,
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Testing and filtering
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Separating and fusing,
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Shortening/lengthening,
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Cutting/linking
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Cloning,
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Synthesizing
Wet-lab and cellular
operations with DNA:
06/11/2013
http://combio.abo.fi/teaching/special
Genetics
Children resemble their
parents:
Although they may look more
like one than the other, most of
them are a blend of the
characteristics of both parents
Useful traits may be
accentuated by controlled
mating:
Speed in horses, strength in
oxes, larger fruits, etc. were
studied in centuries of breeding
of domestic animals and plants
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Question: How are traits
inherited from one generation
to the other?
Story begins with an
Augustinian monk named
Gregor Mendel, 1865
He proposed a “theoretical”
notion of gene responsible for
inheritance
Proposed the three laws of
inheritance
Could not explain what genes
are or where they physically
located
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Chromosomes carry genes
T.H.Morgan, 1904: studies on the physical basis of
heredity:
Organism of choice: fruit fly
Established that genes are physically located on
chromosomes
E.g., the gene for eye color is fruit flies is located on the
X chromosome
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Chromosomes carry genes
Other early examples (for
humans):
Sex-linked genetic
disorders: muscular
dystrophy (1913), red-green
color blindness (1914),
hemophilia (1916)
Disorderss related to
recessive inheritance:
alkaptonuria (1902),
albinism (1903)
Disorders related to
dominant inheritance:
brachydactyly (short fingers,
1905), congenital cataracts
(1906), Huntington’s disease
(1913)
Eye color inheritance (brown
dominant, blue recessive) –
1907
Modern view: more genes
are involved
1941: one gene makes one
protein
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DNA is the genetic material
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Question: Genes are
somewhere on the
chromosomes, but where
are they really placed ?
Chromosomes are
complex structures
consisting of DNA and
many kind of proteins
DNA was discovered about
the same time Mendel and
Darwin published their
work:
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DNA was known to be a
large molecule, but it
seemed likely that its four
chemical components were
assembled in a monotonous
pattern
The hypothesis was that
genes were made of
chromosomal proteins and
DNA was a structural
polymer that was holding the
chromosome together
1941-1952: DNA is indeed
the genetic material
http://combio.abo.fi/teaching/special
DNA is the genetic material
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Question: Genes are
somewhere on the
chromosomes, but where
are they really placed ?
Chromosomes are
complex structures
consisting of DNA and
many kind of proteins
DNA was discovered about
the same time Mendel and
Darwin published their
work:
06/11/2013
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DNA was known to be a
large molecule, but it
seemed likely that its four
chemical components were
assembled in a monotonous
pattern
The hypothesis was that
genes were made of
chromosomal proteins and
DNA was a structural
polymer that was holding the
chromosome together
1941-1952: DNA is indeed
the genetic material
http://combio.abo.fi/teaching/special
Proving that DNA is the
genetic material
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1928 - the Pneumococcus
bacterium causes
pneumonia in mice
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Some strains of
Pneumococcus are highly
virulent (S-bacteria) and
some are non-virulent (Rbacteria)
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Injected in mice, the coat
protects the bacteria from
attack by white blood cells,
they multiply, infect, and
eventually kill the mouse
The presence of the coat is
an inherited genetic trait
A virulent bacteria has a
polysaccharide coat, while
the non-virulent one lacks
the coat
http://combio.abo.fi/teaching/special
Proving that DNA is the
genetic material
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Experiments in 1944 show that genes are made of DNA
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Living S-bacteria injected in mouse: mouse dies
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Living R-bacteria: mouse lives
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Heat-killed S-bacteria: mouse lives
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Heat-killed S-bacteria + living R-bacteria: mouse dies !!!
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Living S-bacteria isolated in the blood of the dead mouse
These bacteria were fully functional, they could replicate and infect
other mice
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The transforming principle
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Conclusion: some chemical process called transforming
principle was responsible for conferring new genetic trait
on non-virulent bacteria
Further experiments: that chemical turned out to be DNA
The concept of DNA as genetic material received
confirmation in another series of experiments in 1952
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Structure of DNA
DNA is composed of building
blocks called nucleotides
A nucleotide consists of a
deoxiribose sugar, a phosphate
group and one of the four
possible bases
The bases are: adenine (A),
thymine (T), guanine (G),
cytosine (C)
Phosphates and sugars of
adjacent nucleotides link
(through strong phosphodiester
bonds) to form a long polymer
The ratio of A-to-T and G-to-C
constant in all living organisms
Final clue provided by X-ray
crystallography: DNA is a
double helix, shaped like a
twisted ladder
Sugar is a pentose (5 carbon
atoms): the phosphate is
attached to the 5’-carbon, the
base to the 1’-carbon
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Structure of DNA
1953: race to describe the 3D structure
won by James Watson and Francis
Crick
Alternate sugar and phosphate molecules
form the twisted uprights of the DNA ladder
The rugs of the ladder are formed by
complementary pairs of bases: A always
paired with T, G always paired with C
One strands runs in the 5’-3’ direction, the
other one in the 3’-5’ direction
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wikipedia
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DNA representation
Single stranded DNA has polarity
It has phosphate (attached to the 5’-carbon of the sugar) available
for binding at one end of the strand and the 3’-carbon of the sugar
available at the other end: the 5’-end and the 3’-end
Four types of nucleotides (A,T,C,G)
One single stranded DNA molecule may be represented as a string
over the alphabet {A,T,C,G}, ALWAYS written in the 5’-to-3’
direction
Example: 5’-ATGCTAC-3’ (most of the time omit 5’ and 3’)
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DNA representation
Representing double stranded DNA molecules: double strings
over the alphabet {A,T,C,G}
Example of one such double string:
5’-ATGTAC-3’
3’-TACATG-5’
Most often we omit the 5’ and 3’
Worth noting that the same molecule is represented also by its
“inverse”
5’-GTACAT-3’
3’-CATGTA-5’
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DNA replication
One half of the DNA ladder serves as a template for
recreating the other half during DNA replication
Responsible for this: the enzyme DNA polymerase that
adds complementary nucleotides to the template provided
by a single stranded DNA molecule
This was conjectured already by Watson and Crick but the
enzyme was actually discovered later
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DNA replication
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Central dogma
DNA mostly found in the nucleus (in eukaryotes)
Another type of nucleic acid commonly found in the cytoplasm:
RNA
RNA copies the DNA message in the nucleus and carries it out
to the cytoplasm, where proteins are synthesized (in the
rybosomes)
DNA transcribed in the nucleus into mRNA
mRNA translated in the cytoplasm (rybosomes) into amino acids
– tRNA plays the role of “adaptor”
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Central dogma
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RNA  protein
From mRNA to protein: the universal genetic code
Each triplet of nucleotides (codon) specifies one amino
acid
One codon specify beginning of translation (AUG) and 3
codons specify the end of it.
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The universal genetic code
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mRNA  protein
Translation mRNA → protein: implemented through some
“adaptors” that recognize both codons and amino acids:
transfer RNA (tRNA)
On one part the tRNA holds an anticodon and on the other
side it holds the corresponding amino acid
Ordering the tRNA molecules on mRNA is complex: using
the ribosome – “large protein synthesizing machine”
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Genes and chromosomes
Each DNA molecule is packaged in a separate
chromosome
The total genetic information stored in the chromosomes:
the genome
Eukaryotes: (almost) every single cell contains a complete
set of the genome
Difference in functionality: different expression of the
corresponding genes
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Coding and non-coding
DNA
Bacteria: virtually all DNA encodes proteins
Eukaryotic DNA is composed of repeated sequences that do not
encode proteins: non-coding sequences (junk DNA)
They separate relatively infrequent “islands” of genes
Many non-coding sequences (introns) are found also within the genes
Less than 5% of the human genome encodes proteins
Human genome: about 3 billion bp
Amoeba: 200 times more DNA than humans
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Chromosomes
Chromsomes are dynamic,
changing structures
DNA may “jump” from one
position on a chromosome to
another (B.McClintock, Nobel
prize)
Genes can be moved between
species based on the universality of
the genetic code
Different bacteria may get antibioticresistant genes by exchanging
plasmids (small chromosomes)
Genes can be turned on and off
Human insulin produced in E.coli
bacteria (1980s)
Regulatory genes produce
inhibitors for other genes
Genetic engineering – exploding
field nowadays
There are thus both genetic
“plans” for protein production
and genetic regulatory
programs for expressing those
plans
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Living things share common
genes
Compelling evidence for shared ancestry of all living things
Evolution of higher life forms require development of new
genes, while retaining the old ones
Genes may be “stolen” from other organisms
Mutations may accumulate – history of the evolutionary life
of a gene
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Beyond DNA
Passive role for DNA: encodes and transmits genetic info
through time
Active role for proteins encoded by DNA
Post-genomic phase: what is the exact role of the proteins
made by these specific genes?
Animal of choice for test: mice
They share about 99% of the genes with humans
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A classification
Living organisms:
Prokaryotes: single-cell organisms with no nucleus (e.g., simple
bacteria)
Eukaryotes: DNA protected in a nucleus, genes splitted in exons
and introns (e.g., humans)
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Wet-lab methods to
manipulate DNA
We will overview the methods used in the lab and in the cell
itself to manipulate DNA
The availability of such tools made the rapid development of the field
possible
It is not a lab manual, only present a number of available tools and the
ideas supporting them
Essential to know about elementary lab tools also for mathematicians and
computer scientists
Topics: measuring DNA, testing for/isolating DNA molecules,
separating and fusing DNA strands, lengthening, shortening,
cutting, linking DNA, modifying nucleotides, multiplying,
sequencing
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Measuring the length of
DNA molecules
Problem: measure the length of a molecule without
sequencing it
Length – definition:
Single stranded DNA: the number of nucleotides (ex: 12 mer)
Double stranded DNA: the number of base pairs (ex: 12 bp)
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Measuring the length of
DNA
Central fact: DNA molecules are negatively charged: in
electric fields, they migrate towards + electrodes (anodes)
The force needed to move DNA molecules – proportional
to its length (due to friction)
Idea: gel electrophoresis technique
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The gel electrophoresis
technique
Pour some gel in a rectangular container with electrodes
on the sides
In the cooling process: a comb is inserted on the negative
side
Gel cooled down, comb removed: row of small wells
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The gel electrophoresis
technique
The solution with the DNA to be measured brought in the wells
Activate the electric field: DNA molecules travel towards the
anode
Gel acts as a molecular sieve – big friction
small molecules move faster than big molecules
Deactivate the field when the first molecules have reached the anode
Small molecules – longer distance than bigger molecules
Molecules of same length – same distance
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The gel electrophoresis
technique
Reading the resulting gel
DNA molecules are colorless: mark them
Two techniques for marking:
Staining with ethridium bromide -> fluorescent mark under ultraviolet light
Attaching radioactive markers to the ends of DNA molecules -> expose a
film
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Computing the length
Based on the distance traveled
Using a calibrating solution in one of the wells -> compare
the location of bands on the other paths with the calibrating
path
Digital printout of an agarose gel
electrophoresis of cat-insert plasmid DNA
- Wikipedia
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DNA gel electrophoresis - Wikipedia
http://combio.abo.fi/teaching/special
Gels of plasmid preparations usually show a
major band of supercoiled DNA with other
fainter bands in the same lane. Note that by
convention DNA gel is displayed with smaller
DNA fragments near the bottom of the gel.
This is because historically DNA were run
vertically and the smaller DNA fragments
move downwards faster. - Wikipedia
Testing for/isolating known
molecules
Problem: test if some known single stranded molecules s exist
in a given DNA solution and isolate some of them if so
Solutions:
Attach the complementary strands (probes) to a filter and pour the solution
through the filter
Attach probes to tiny glass beads, place them in a glass column and pour
the solution over them
Attach probes to tiny magnetic beads, throw them in the solution and stir;
attract them to one side using a magnet
Result:
s molecules bind to their complements to form double strands (annealing)
and stay there
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DNA microarray
Basic function: detect and
quantify DNA subsequences
in a sample collection of DNA
molecules
Basic principle:
chip (either glass, plastic or
silicon) contains thousands of
DNA spots organized in an
ordered uniform manner (array)
Each DNA spot contains
picomoles of copies of short
sDNA sequences (probes)
The probe DNA sequence is
associated to a specific position
on the chip
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Measurement:
Prepare sample, label
fluorescent target DNA
sequences
Let target sequences to
hybridize with the
complementary probes
Wash-out weakly hybridized
target DNAs
Result:
Only DNAs with the
subsequences matching the
probes will stay bound to the
corresponding DNA spots on
the chip
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DNA microarray
Analysis:
Fluorescently labeled DNA targets bound to probes produce light signal. More
targets bound to a spot → higher the signal
Scan the microarray image, basing on the position of a spot and its signal level –
quantify occurance of DNA subsequences in the sample
Issues:
Denoising
normalization
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Wikipedia
http://combio.abo.fi/teaching/special
DNA microarray
Usage:
Gene expression profiling
Comparative genomic hybridization
Gene identification – test for hallmarks of microorganisms in food and feed
Detect protein-DNA binding sites
SNP detection
Alternative splicing and fusion genes detection
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Separating and fusing DNA
strands
Hydrogen bond between complementary bases is weaker
than the covalent bond between consecutive nucleotides
within one strand
One can separate the two strands of a DNA molecule
without breaking the single strands
Separating the two strands: denaturation - heating
Fusing two complementary strands:
annealing/renaturation/hybridization - cooling
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Enzymes
Enzymes: proteins that catalyze biochemical reactions
Very specific: most catalyze just a single chemical reaction
Highly efficient: speed up the chemical reaction by as much as a
trillion times
If no enzymes existed, bio-reactions would most likely be
too slow to support life
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Lengthening DNA polymerases
The enzymes: polymerases - add
nucleotides to an existing DNA molecule
Polymerases only extend the DNA
molecules in the 5’-3’ direction
They extend repeatedly the 3’ end,
provided that the required nucleotides
are available in the vicinity
Given one strand, to produce the
corresponding double stranded molecule,
one needs the primer and the
polymerases
Polymerase in action
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Lengthening DNA
terminal transferase does not
need a template: extends
double strands at both ends
by some single stranded tails
Engineering single strands is
possible: a procedure leading
to automation exists →
“synthesizing robots”
Short such single strands are
called oligonucleotides (oligos
in short); they are essential in
PCR
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Shortening/cutting DNA
The enzymes: DNA
nucleases – degrade
DNA
Two types of enzymes:
DNA exonucleases:
cleave (remove) one
nucleotide at a time from
the ends of the DNA
DNA endonucleases:
destroy internal bonds in
the DNA molecule (cutting
DNA)
Restriction
endonucleases – cut on
the specific sites
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endonuclease
exonuclease
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Linking DNA
Fuse together two pieces of
DNA
Add the necessary chemical
links
Examples
Hydrogen bonding
Ligation (enzyme: ligase)
Hybridization: link two
complementary single strands
Blunt end ligation
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Multiplying DNA
Major problem in biochemical
research: obtain sufficient quantities
of some substance
Solution: cloning techniques, PCR
Cloning in vivo:
insert the DNA fragment in the DNA
of some fast reproducing cell and
the fragment will be replicated with
the cell
Cloning in vitro:
Polymerase chain reaction – PCR
1985, Kary Mullis: Nobel prize
Very efficient: produce in a short
time billions of copies of even a
single strand
The PCR technique:
Use viruses or hybridization
This technique provides high
quantities of DNA fragments
It also gives a mean to preserve the
fragments for long time (keeping
alive the “host”)
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We want to amplify a DNA
molecule alpha with known borders
beta and gamma
Process: repeat a cycle of
denaturation, priming, extension
Start: prepare a solution containing
alpha (the target), oligonucleotides
beta’ and gamma’ (complements of
beta and gamma), polymerase
enzymes, and many nucleotides
(A, C, G, T)
The PCR technique
Denaturation: heat the solution
to 85-95 C: alpha denatures into
two single strands alpha1 and
alpha2
Priming: Cool down the solution
to 55C: the primers beta’ and
gamma’ anneal to their
complementary borders
denaturation
Extension: Heat the solution to
72C: polymerase extend the
primers to produce two double
stranded DNA molecules alpha
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priming
extension
The PCR technique
Repeat the cycle n times: 2n copies (in principle): highly
efficient bio-copymachine!
A single cycle takes about 5 minutes: obtain billions of
copies in several hours (days for cloning)
The polymerase must be heat resistant – nature’s solution:
thermophilic bacteria
Important observation:
one needs to know the borders of the DNA segment to be copied
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Sequencing
Learning the exact sequence of nucleotides of a DNA molecule
The most popular method of sequencing: the Sanger method
1940s: Sanger, insulin sequencing (Nobel prize)
1977: Sanger, Gilbert, DNA sequencing (Nobel prize)
1985: Mullis, PCR (Nobel prize)
Main tool: nucleotide analogues – nucleotides chemically altered
so that no other nucleotide can attach to their 3’ end: ddA, ddC,
ddG, ddT (dideoxynucleotides)
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Sequencing – Sanger
method
Problem: sequence a single
stranded molecule alpha
If only nucleotides are used:
produce full duplex
Extend it to 3’ by gamma and
add the primer gamma’
(labeled): let beta be the new
molecule
Solution: tube X contains a limited
amount of nucleotide analogues
ddX, for all X in the set {A,C,T,G}
Prepare 4 tubes (A,C,G,T)
containing:
Beta molecule – ready to sequence
beta molecules
Primers gamma’
Nucleotides
Incomplete molecules in tube A
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Example: alpha=3’-AGTACGTGACGC
Tube A:
Tube C:
5’- gamma’TCATGCACTGCG
5’- gamma’TCATGCACTGCG
5’- gamma’TCA
5’- gamma’TC
5’- gamma’TCATGCA
5’- gamma’TCATGC
5’- gamma’TCATGCAC
5’- gamma’TCATGCACTGC
Tube G:
Tube T:
5’-gamma’TCATGCACTGCG
5’- gamma’T
5’- gamma’TCATGCACTGCG
5’- gamma’TCATG
5’- gamma’TCATGCACTG
5’- gamma’TCAT
5’- gamma’ TCATGCACT
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Sequencing – Sanger
method
Gel electrophoresis using 4 wells - one for each tube
Read the bands (the primers were marked)
Obtain the sequence
Sequencing ladder
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Sanger method,
observations
This was only a very simplified presentation
Use polymerase not having the associated exonuclease activity: do not cut
the ddX
Limited amount of nucleotide analogues: too many make the polymerase
before then end of the strand
Impossible to sequence long molecules with this method
Sequencing a longer molecule: divide it in overlapping short segments,
sequence them and then reassemble the original sequence:
computational (math) problem!
More recently, Sanger sequencing has been supplanted by "Next-Gen"
sequencing methods, especially for large-scale, automated genome
analyses. However, the Sanger method remains in wide use, primarily
for smaller-scale projects and for obtaining especially long contiguous
DNA sequence reads (>500 nucleotides).
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NextGen sequencing
The high demand for low-cost sequencing has driven the
development of high-throughput sequencing
NextGen sequencing produces thousands or millions of
sequences concurrently.
High-throughput sequencing technologies are intended to lower
the cost of DNA sequencing beyond what is possible with
standard dye-terminator methods.
In ultra-high-throughput sequencing as many as 500,000
sequencing-by-synthesis operations may be run in parallel.
e.g., Illumina (Solexa) sequencing
e.g., SOLiD sequencing
Faster >1000 times, cheaper <1000 times
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Acknowledgements
Ion Petre's course “Introduction to
Biocomputing, Fall 2004”
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