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Major Groove
Minor Groove
T A
G C
T T
T
G
G
G
G
A
A
A
C
C
C C
5' 3'
3' 5'
0.34 nm between nucleotides
One helical turn=
3.4 nm
10 nucleotides
DNA Structure
3'
5' 3'
5'
Anti-parallel orientation
A. The Concept
DNA has a regular structure. It's orientation, width, width between nucleotides, length and number of nucleotides per helical turn is constant. All of these features were described by Watson and Crick. Adenine is always opposite thymine, and cytosine is always oppostie guanine. The two strands are held to- gether by hydrogen bonds: two bonds between adeninine and thymine and three bonds between guanine and cytosine.
2.0 nm Helix Nucleotides Helix Form Direction per turn Diameter A Right 11 2.3 nm B Right 10 2.0 nm Z Left 12 1.8 nm
Figure 3. The structure of common DNA molecules.
..
.. .
.. .
.. .
..
. (^). . ..
..
.. .
This figure describes the general features of B DNA , the most common structure found within a cell. Other forms of DNA also exist. All forms have unique features. These are:
Deoxyribonucleotide Structure
A. The Concept
DNA is a string of deoxyribonucleotides. These consist of three different components. These are the dexoyribose sugar , a phosphate group , and a nitrogen base. Variation in the nitrogen base composition distingushes each of the four deoxyribonucleotides.
The basic building block is the deoxyribose sugar. This sugar is distinguished because it contains a hydrogen (H) atom at the number 2' carbon. Normal ribose has a hydorxyl (-OH) group at this position.
Attached to the 5' carbon is a triphosphate group. This group is important because in a DNA chain it undergoes a reaction with the 3' OH group to produce polydeoxynucleotide.
The final feature of the molecule is a nitrogen base. These are attached to the 1' carbon. Four bases are possilbe. Two pyrimidines (thymie and cytosine) and two purines (adenine and guanine). The double stranded DNA molecule is held together by hyrodgen bonds. Pairing involves specific atoms in each base. Adenine pairs with the thymine, and guanine pairs with cytosine. These pairings and the atoms involved are shown to the right.
You have probaly heard of ATP, the energy moleucle. It is the deoxyribonucleotide to which adenine is attached. This molecule serves two very important functions in biological organisms.
Nitrogen Bases Pyrimidines
C C CH N
C
N
CH
O
O
H 2 (^21)
3 45 6
Thymine
C CH CH N
C
N
O
N
(^21)
3 45 6
Cytosine
HC
N
N
C C C N
C
NH N
O
1 4 3 2 87 5 6 9
Purines
HC
N
N
C C CH N
C
N
N
1 4 3 2
87 5 6 9
Adenine
Guanine
H H
H H
H H
C
C C
C
O
CH
H
OH H
H
2
O
O
O
O O O
O
O
P O P O P
1' 3' 2'
4'
γ β α 5'
Nitrogen
Base
Phosphate Group
Sugar Moiety
Basic deoxyribonucleotide components
Figure 4. The structure of deoxyribonucleotides and base pairing among N bases.
Making a Phosphodiester Bond/
Growing the DNA Chain
C C C
C
CH O
H H
H
2
O O
O
O O O
O
O P O P O P 1' 3' 2'
4'
γ β α^ 5'^ Nitrogen Base
C C C
C
CH O
H H
H
O 2
O
O
O P 1' 3' 2'
4'
α 5'^ Nitrogen Base
C C C
C
CH O
H OH H
H
O 2
O
O
O P 1' 3' 2'
4'
α 5' Nitrogen Base
5' end
3' end
C C C
C
CH O
H H
H
2
O O
O
O O O
O
O P O P O P 1' 3' 2'
4'
γ β α^ 5'^ Nitrogen Base
OH
O C C C
C
CH O
H H
H
2
O O
O
O O O
O P O P O P 1' 3' 2'
4'
γ β α 5'^ Nitrogen Base
C C C
C
CH O
H H
H
O 2
O
O
O P 1' 3' 2'
4'
α 5'^ Nitrogen Base
C C C
C
CH O
H H
H
O 2
O
O
O P 1' 3' 2'
4'
α 5' Nitrogen Base
5' end
3' end
C C C
C
CH O
H H
H
O 2
O
O
P 1' 3' 2'
4'
α^ 5'^ Nitrogen Base
OH
O
O
O O
O
O Pγ O Pβ O
(Pyrophosphate)
A. The Concept
The addition of a new nucleotide to a DNA molcule creates a phosphodiester bond. This requires the DNA chain that is being elongated and a deoxyribo- nucleotide.
Phosphodiester bonds are formed when a news dideoxynucleotide is added to a growing DNA molecule. During the reaction, a condensation reaction occurs between the α phosphate of the nucleotide and the hyroxyl group attached to the 3' carbon. This reaction is performed by the enzyme DNA polymerase. This is also an energy requiring reaction. The energy is provided by the breaking of the high-energy phophate bond in the nucleotide. This results in the release of a pyrophosphate molecule.
Figure 6. The formation of the phosphodiester bond that grows the DNA chain.
Steps of DNA Replication
(Part 1)
3. An error occurs during DNA replication. 4. The DNA replication error is removed by 3'-5' exonuclease function of DNA polymerase.
5' 5'
3' 3' 5'
ATTGAT
Replication Error Removed
5' 5'
3' 3' 5'
ATTGAT
TAACTT
Replication Error
1. The replication fork is formed; RNA primer added. 2. DNA is replicated by the 5'-3' synthesis function of DNA polymerase using the leading strand in a continuous manner.
5' 5'
3' 3'
5'
Leading Strand OH-3'
Continuous Replication
Replication Fork
5' 5'
3' 3'
5' (^) OH-3'
RNA Primer
OH-3'
A. The Concept
DNA replication is essential biological process. It's primary function is to produce new DNA for cell division. The process has several distinct steps that are important to understand. The factors that are absolute requirements for DNA replication to begin are a free 3'-OH group and a DNA template. A RNA primer provides the free 3'-OH group. The DNA to be replicated serves as the template. It is important to remember that all DNA replication proceeds in the 5'-3' direction.
Notes on E. coli replication : DNA Polymerase I and III. Pol III is the primary replicase enzyme that performs the elongation of the DNA strand. It adds nucleotides first to the RNA primer and then grows the chain by creating the phosphodiester bonds. It also has a 3'-5' proofreading (exonulcease) function that removes incorreclty incorporated nucleotides. DNA Pol I also has the 5'-3' replicase function, but it is primarily used to fill the gaps in the replicated DNA that occur when the RNA primer is removed. This enzyme also has a 5'-3' exonuclease function that is used to remove the RNA primer.
Figure 7. The steps of DNA replication.
Chain Termination Sequencing:
the Sanger Technique
When a dideoxynucleotide is inserted, the DNA replication process terminates because dideoxynucleotides do not have the necessary free 3' hydroxyl group required for the addition of additional nucleotides. This results in fragments that differ by one nucleotide in length.
C C C
C
CH O
H H H
H
2
O O
O
O O O
O
O P O P O P 1' 3' 2'
4'
γ β α 5' Nitrogen Base
Phosphate Group
Sugar Moiety
Note: neither the 2' or 3'carbon has an OH group
a. A dideoxynucleotide b. The reaction reagents
DNA template sequencing primer dNTPs ddNTPs (low concentration) DNA polymerase salts
c. The sequencing reaction result: fragments that differ
by one nucleotide in length
A T T C G G A T C C T T A A 5' T A A G C C T A G G A A T T - H 3' 5' T A A G C C T A G G A A T - H 3' 5' T A A G C C T A G G A A - H 3' 5' T A A G C C T A G G A - H 3' 5' T A A G C C T A G G - H 3' 5' T A A G C C T A G - H 3' 5' T A A G C C T A - H 3' 5' T A A G C C T - H 3' 5' T A A G C C - H 3' 5' T A A G C - H 3' 5' T A A G - H 3' 5' T A A - H 3' 5' T A - H 3' 5' T - H 3'
Template Primer
A. The Concept
DNA sequencing is the most techique of genomics. By collecting the sequence of genes and genomes we begin to understand the raw material of phenotype devel- opment. The most common DNA sequencing is called chain termination sequencing or the Sanger technique (named after the person who created it). It is called chain termination because the incorporation of a dideoxynucleotide terminates the replication process because the nucleotide lacks the required 3'-OH group.
Figure 8. The chain termination (Sanger) DNA sequencing technique.
A T T C G G A T C C T T A A 5' T A A G C C T A G G A A - H 3' 5' T A A G C C T A G G A - H 3' 5' T A A G C C T A - H 3' 5' T A A - H 3' 5' T A - H 3'
Gel-based Detection of DNA Sequences
A. The concept
Four DNA sequencing reactions are performed. Each contains only one of the four dideoxynucleotides. Each reaction is added to a single lane on the gel. Since one of the dNTPs is radioactive, the gel in which the fragments are separated, can be used to expose an x-ray film and read the sequence.
a. The sequencing products
Reaction with ddATP
A T T C G G A T C C T T A A 5' T A A G C C T A G G A A T T - H 3' 5' T A A G C C T A G G A A T - H 3' 5' T A A G C C T - H 3' 5' T - H 3'
A T T C G G A T C C T T A A 5' T A A G C C T A G G - H 3' 5' T A A G C C T A G - H 3' 5' T A A G - H 3'
A T T C G G A T C C T T A A 5' T A A G C C - H 3' 5' T A A G C - H 3'
Reaction with ddTTP
Reaction with ddGTP
Reaction with ddCTP
b. The sequencing gel
The sequencing reactions are separated on a polyacrylamide gel. This gel separates the fragments based on size. The shorter fragments run further, the longer fragments run a shorter distance. This allows the scientists to read the sequence in the 5'-3' direction going from the bottom to the top of the gel.
T
T
A
T
A
A
G
C
C
T
G
G
G
A
5'
3'
G A T C
Figure 9. Gel-based detection of DNA sequencing products.
A. The Concept
Hierarchical shotgun sequencing requires that large insert libraries be constructed. A series of these clones are ordered by several techniques. Once these clones are ordered, each clone is separately fractionated into small fragments and cloned into plasmid vectors. The plasmid clones are sequenced, and the sequence is assembled. This is the procedure used to sequence the Arabidoposis genome, and by the public project to sequence the human genome.
Large insert libraries of nuclear DNA are created in BAC, PAC or YAC vectors.
Large inserts clones are placed in order usng hybridization, fingerprinting, and end sequencing. This tiling path consists of individual large insert clones that will be sequenced.
Each ordered clone is fractionated into small fragments and cloned into a plasmid vector. This is called shotgun cloning.
Each clone is then end-sequenced , and the sequences of all the clones are aligned.
A T T C G T T A G C G A T T A A G C G A T T A T T A G A T A C
Hieracrchical Shotgun Sequencing
of Genomes
A. The Concept
Shotgun sequencing requires that random, small insert libraries are created from the total nuclear DNA of the species of interest. A plasmid cloning vector is used for this step. These clones are then sequenced. This step is analogous to the shotgun cloning and sequencing step used for each large-insert clone used in hierarchial shotgun. The sequences of the clones are then aligned. This is the procedure used to sequence the Drosophila genome, and by Celera to sequence the human genome.
Nuclear DNA is fractionated into several size classes. The most abundant class used for clone creation is 2kb in size. Large inserts (10- 50 kb) are also used.
Each clone is then end-sequenced from both ends to develop read pairs or mate pairs. The sequences of all the clones are aligned. A T T C G T T A G C G A T T A A G C G A T T A T T A G A T A C
Whole Genome Shotgun Sequencing
The fragments are then cloned into a plasmid vector.
Hierarchical Shotgun Sequencing
Two major sequencing approaches o Hierarchical shotgun sequencing o Whole genome shotgun sequencing Hierarchical shotgun sequencing o Historically First approach o Why??? Techniques for high-throughput sequencing not developed Sophisticated sequence assembly software not availability Concept of the approach o Necessary to carefully develop physical map of overlapping clones Clone-based contig ( contig uous sequence) o Assembly of final genomic sequence easier o Contig provides fixed sequence reference point But o Advent of sophisticated software permitted Assembly of a large collection of unordered small, random sequence reads might be possible o Lead to Whole Genome Shotgun approach
Steps Of Hierarchical Shotgun Sequencing
Requires large insert library BAC or P1 (bacterial artificial chromosomes) Primary advantages o Contained reasonable amounts of DNA about 75-150 kb (100,000 – 200,000) bases o Do not undergo rearrangements (like YACs) o Could be handled using standard bacterial procedures
Developing The Ordered Array of Clones for Sequencing
Using a Molecular Map o DNA markers o Aligned in the correct order along a chromosome o Genetic terminology Each chromosome is defined as a linkage group o Map: Is reference point to begin ordering the clones Provides first look at sequence organization of the genome Overlapping the clones o Maps not dense enough to provide overlap o Fingerprinting clones Cut each with a restriction enzyme ( Hin dIII) Pattern is generally unique for each clone Overlapping clones defined by Partially share fingerprint fragments o Overlapping define the physical map of the genome
Sequencing Clones of The Minimal Tiling Path
Steps o Physically fractionate clone in small pieces o Add restriction-site adaptors and clone DNA Allows insertion into cloning vectors Plasmids current choice o Sequence data can be collected from both ends of insert Read pairs or mate pairs Sequence data from both ends of insert DNA Simplifies assembly Sequences are known to reside near each other
Assembly of Hierarchical Shotgun Sequence Data
Process o Data collected o Analyzed using computer algorithms o Overlaps in data looked for
Confirming the Sequence
Molecular map data o Molecular markers should be in proper location Fingerprint data o Fragment sizes should readily recognized in sequence data
Whole Genome Shotgun Sequencing (WGS)
Hierarchical sequencing approach o Begins with the physical map o Overlapping clones are shotgun cloned and sequenced WGS o Bypasses the mapping step Basic approach o Take nuclear DNA o Shear the DNA o Modify DNA by adding restriction site adaptors o Clone into plasmids Plasmids are then directly sequenced Approach requires read-pairs Especially true because of the repetitive nature of complex genomes
WGS
Proven very successful for nearly all sized genomes o Essentially the only approach used to sequence smaller genomes like bacteria Early question: Is WGS useful for large, complex genomes? o Initially consider a bold suggestion o Large public effort dedicated to hierarchical approach o Drosophila Sequenced using the WGS approach o Rice Two different rice genomes sequenced using WGS approach
Pyrosequencing in Picolitre Reactors
Pyrosequencing reagents
DNA template (DNAn) DNA polymerase A dideoxynucleotide o dNTP o deoxyadenosine thio triphospate substitutes for dATP ATP sulfurlyase Adenosine 5’ phosphosulfate (APS) Luciferase Luciferin Apyrase
Pyrosequencing reactions
DNA Polymerase
(1) DNAn + a dNTP DNAn+1 + PPi
ATP Sulfurylase
(2) PPi + APS ATP
Luciferase
(3) ATP + Luciferin Oxyluciferin + Light
Apyrase
(4) dNTP dNDP + dNMP + phosphate
Apyrase
(5) ATP dADP + dAMP + phosphate
