Nucleic Acid Basics• Nucleic Acids Are Polymers• Each Monomer Consists of Three Moieties:
Nucleotide
A Base + A Ribose Sugar + A PhosphateNucleoside
• A Base Can be One of the Five Rings:
• Unlike three dimensional structures of proteins, DNA molecules assume simple double helical structures independent of their sequences. There are three kinds of double helices that have been observed in DNA: type A, type B, and type Z, which differ in their geometries. The double helical structure is essential to the coding function of DNA. Watson (biologist) and Crick (physicist) first discovered the double helix structure in 1953 by X-ray crystallography.
• RNA, on the other hand, can have as diverse structures as proteins, as well as simple double helix of type A. The ability of being both informational and diverse in structure suggests that RNA was the prebiotic molecule that could function in both replication and catalysis (The RNA World Hypothesis). In fact, some viruses encode their genetic materials by RNA (retrovirus)
Forces That Stabilize Nucleic Acid Double Helix
• There are two major forces that contribute to stability of helix formation– Hydrogen bonding in base-pairing– Hydrophobic interactions in base stacking
5’ 3’
3’
Same strand stacking
cross-strand stacking
5’
Types of DNA Double Helix
• Type A: major conformation of RNA, minor conformation of DNA;
• Type B: major conformation of DNA;• Type Z: minor conformation of DNA
5’ 3’
3’
5’
5’
3’
3’
5’
5’
3’
3’ A BNarrow tight
Wide Less tight
Left-handedLeast tight
Z5’
Secondary Structures of Nucleic Acids
• DNA is primarily in duplex form.
• RNA is normally single stranded which can have a diverse form of secondary structures other than duplex.
More Secondary Structures of Nucleic Acids
Pseudoknots:
Source: Cornelis W. A. Pleij in Gesteland, R. F. and Atkins, J. F. (1993) THE RNA WORLD. Cold Spring Harbor Laboratory Press.
3D Structures of RNA: Transfer RNA Structures
Tertiary Structureof tRNA
Secondary Structureof tRNA
AnticodonStem
D Loop
TψC Loop
Variableloop
Anticodon Loop
3D Structures of RNA: Ribosomal RNA Structures
Secondary StructureOf large ribosomal RNA
rRNA Secondary Structure Based on Phylogenetic Data
Tertiary StructureOf large ribosome subunit
Ban et al., Science 289 (905-920), 2000
DNA Sequencing
Chain Termination Method– Sanger, 1977– single stranded DNA, ~800b– Method:
• Electrophoresis can separate DNA molecules differing 1bp in length
• Dideoxynucleotide (ddNTP) are used - which stop replication
ddNucleotides
ddA, ddT, ddC, ddGEach type marked with fluorescent dyeWhen incorporated into DNA chain –stops replication
Chain Termination Method, An Outline
Replication– Obtaining ssDNA– Add a (universal) primer
Start replication in a soup of A,T,C,G
Continously add tiny amounts of ddA, ddT, ddC, ddG– gradually stopping all the processes
Chain Termination Method,Reading the Sequence
Running through electrophoresis gel– Four types of ddNTP have four
different fluorescent labels– Automated reading
See: http://www.dnalc.org/Shockwave/cycseq.html
Chain Termination Method,Results
Electrophoresis and laser beam scanning
Sign
al
timefragment sizeElectropherogram
Shotgun Method - OverviewCut genome into short fragmentsSequence DNA fragmentsCreate contigs
Contig - continous set of overlapping sequences
Gap!
Shotgun Method
The shotgun approach to sequence assembly. The DNA molecule is broken into small fragments, each of which is sequenced. The master sequence is assembled by searching for overlaps between the sequences of individual fragments. In practice, an overlap of several tens of base pairs would be needed to establish that two sequences should be linked together.
Shotgun Method –Contig Construction
Two DNA sequences:X=CTATCAY=AGTATHow do they overlap?
Try to apply dynamic programmingorX XY Y
Shotgun Method –Haemophilus Influenzae Sequencing
1.5-2kb
Extract DNA
Sequence Construct contigs
Sequenced
SonicateDNA library
Electrophoresis
Shotgun Method - Filling in gapsContig Contig
Probe libraries
GapContig
Gap
Scaffold A series of sequence contigs separated by sequence gaps.
Shotgun Method - Pros and Cons
Pros– Human labour reduced to minimum
Cons– Computationally demanding – O(n2)
comparisons– High error rate in contig construction
• Repeats as the main problem
Shotgun vs. Hierarchical Method
Celera vs. Human Genome ProjectHierarchical (top-down) assembly:– The genome is carefully mapped– “Shotgun” into large chunks of 150kb
• Exact location of each chunk is known– Each piece is again “shotgunned” into 2kb
and sequenced
New Sequencing Methods
Sequencing By Hybridization– Check which from all possible fragments of length
k (k-tuples) hybridize to the sequence
TAA ATTCGTAAAAGAGCAAG
AGC