3.
1o Structure - Linear array of nucleotides
2o Structure – double helix
3o Structure - Super-coiling, stem-loop
formation
4o Structure – Packaging into chromatin
4.
Can determine the sequence of DNA base pairs in
any DNA molecule
Chain-termination method developed by Sanger
Involves in vitro replication of target DNA
Technology led to the sequencing of the human
genome
5. DNA Replication
• DNA is a double-helical molecule
• Each strand of the helix must be copied in
complementary fashion by DNA polymerase
• Each strand is a template for copying
• DNA polymerase requires template and
primer
6.
Primer: an oligonucleotide that pairs with
the end of the template molecule to form
dsDNA
DNA polymerases add nucleotides in 5'-3'
direction
7.
8. Chain Termination Method
• Based on DNA polymerase reaction
• 4 separate rxns
• Each reaction mixture contains dATP, dGTP,
dCTP and dTTP
9.
Each reaction also contains a small amount of
one dideoxynucleotide (ddATP, ddGTP, ddCTP
and ddTTP).
Each of the 4 dideoxynucleotides are labeled
with a different fluorescent dye.
Dideoxynucleotides missing 3’-OH group. Once
incorporated into the DNA chain, chain
elongation stops)
10. Chain Termination Method
• Most of the time, the polymerase uses
normal nucleotides and DNA molecules grow
normally
• Occasionally, the polymerase uses a
dideoxynucleotide, which adds to the chain
and then prevents further growth in that
molecule
11.
Random insertion of dd-nucleotides leaves
(optimally) at least a few chains terminated
at every occurrence of a given nucleotide
14. Chain Termination Method
• Run each reaction mixture on electrophoresis gel
• Short fragments go to bottom, long fragments on
top
• Read the "sequence" from bottom of gel to top
• Convert this "sequence" to the complementary
sequence
• Now read from the other end and you have the
sequence you wanted - read 5' to 3'
15.
16.
17.
DNA is double stranded with antiparallel
strands
Right hand double helix
Three different helical forms (A, B and Z
DNA.
18. Comparison of A, B, Z DNA
• A: right-handed, short and broad, 2.3 A, 11
bp per turn
• B: right-handed, longer, thinner, 3.32 A, 10
bp per turn
• Z: left-handed, longest, thinnest, 3.8 A, 12
bp per turn
20. Z-DNA
• Found in G:C-rich
regions of DNA
• G goes to syn
conformation
• C stays anti but
whole C nucleoside
(base and sugar)
flips 180 degrees
21.
Double Strand DNA can be denatured by
heat (get strand separation)
Can determine degree of denturation by
measuring absorbance at 260 nm.
22.
Conjugated double bonds in bases absorb
light at 260 nm.
Base stacking causes less absorbance.
Increased single strandedness causes
increase in absorbance
23.
24.
Melting temperature related to G:C and
A:T content.
3 H-bonds of G:C pair require higher
temperatures to denture than 2 H-bonds of
A:T pair.
27. Supercoils
• In duplex DNA, ten bp per turn of helix
(relaxed form)
• DNA helix can be over-wound.
• Over winding of DNA helix can be
compensated by supercoiling
28.
Supercoiling prevalent in circular DNA
molecules and within local regions of long
linear DNA strands
Enzymes called topoisomerases or gyrases can
introduce or remove supercoils
In vivo most DNA is negatively supercoiled.
Therefore, it is easy to unwind short regions of
the molecule to allow access for enzymes
29. Each super coil compensates for one + or –
turn of the double helix
30. Cruciforms occur in palindromic regions
of DNA
Can form intrachain base pairing
Negative supercoiling may promote
cruciforms
34. Chromosome Structure
• Human DNA’s total length is ~2 meters!
• This must be packaged into a nucleus that is
about 5 micrometers in diameter
• This represents a compression of more than
100,000!
35.
It is made possible by wrapping the DNA
around protein spools called nucleosomes
and then packing these in helical filaments
36. Nucleosome Structure
• Chromatin, the nucleoprotein complex,
consists of histones and nonhistone
chromosomal proteins
• % major histone proteins: H1, H2A, H2B,
H3 and H4
37.
Histone octamers are major part of the
“protein spools”
Nonhistone proteins are regulators of gene
expression
38. •4 major histone (H2A, H2B, H3, H4) proteins for
octomer
•200 base pair long DNA strand winds around the
octomer
•146 base pair DNA “spacer separates individual
nucleosomes
•H1 protein involved in higher-order chromatin
structure.
•W/O H1, Chromatin looks like beads on string
42.
Single stranded molecule
Chemically less stable than DNA
presence of 2’-OH makes RNA more
susceptible to hydrolytic attack (especially form
bases)
Prone to degradation by Ribonucleases (Rnases)
43.
Has secondary structure. Can form
intrachain base pairing (i.e.cruciform
structures).
Multiple functions
44.
Ribosomal RNA (rRNA) – integral part of
ribosomes (very abundant)
Transfer RNA (tRNA) – carries activated amino
acids to ribosomes.
45.
Messenger RNA (mRNA) – endcodes
sequences of amino acids in proteins.
Catalytic RNA (Ribozymes) – catalzye
cleavage of specific RNA species.
