DNA Structure
The deoxyribonucleic acid, DNA, is a long chain of
nucleotides which consist of
•
Deoxyribose (a pentose = sugar with 5 carbons)
•
Phosphoric Acid
•
Organic (nitrogenous) bases (Purines - Adenine and
Guanine, or Pyrimidines -Cytosine and Thymine)
Bases
Purine (A+G)
Pyrimidine (T+C)
a nucleotide, a building block of DNA. It is a phosphate ester of a nucleoside
Nucleotide
Nucleoside
a nucleotide, a building block of DNA
Note numbering
system for carbons
in ring
Also note the
difference between
a ribose used for
building DNA and
one used for RNA
DNA and RNA chains are
made by connecting
nucleotides together via
chemical bonds
What is this chain RNA or
DNA?
• four different types of nucleotide possible
in a DNA sequence, adenine, cytosine,
guanine and thymine (ATCG)
• Nucleotides are situated in adjacent pairs
in the double helix.
• Thymine and adenine can only make up a
base pair
• Guanine and cytosine can only make up a
base pair
• Double-stranded DNA
is simply two chains of
single- stranded DNA,
positioned so their
"bases" can interact
with each other.
• The sugar-andphosphate 'backbone'
is red, and the bases
are blue.
• the two strands travel
in opposite directions;
"anti-parallel".
• The bases in the
middle "pair up" with
bases on the opposite
strand, A+T, G+C
• Hydrogen bonds hold
stucture together
Genome- entire complement of genetic
information.
•
Includes coding and non coding
•
Genes (exons and introns)
– Alleles are different gene forms
•
Useful DNA for doing genome analysis
Plasmid- extra chromosomal DNA, found naturally
mainly in bacteria
•
covalent closed circle, double stranded DNA
•
Non essential
•
Replicates independently
•
Occurs naturally in bacteria,
•
Molecular biologists recognise use and made them
their own
•
Used as cloning vectors i.e. to transfer DNA
between bacteria
Recombinant plasmids made by molecular biologists
have been designed to carry foreign DNA into bacterial
cells.
They have
•
unique restriction enzyme sites. Usually many different
ones in a polylinker site
•
origin of replication for bacteria
•
selectable marker (often antibiotic resistance)
DNA isolation and purification
•
•
Important to obtain clean intact DNA in sufficient quantities to
work with
Always do on ice
Most purification procedures include many of the following steps
1.
2.
3.
4.
Lysis of cells to release contents including DNA
Treatment with EDTA to bind divalent cations
Proteinase K treatment to digest proteins and tissue away from
DNA
Separation of DNA from other contaminants in cellular soup using
chemical and physical differences e.g. differential solubilities,
precipitation, binding to columns and centrifugation
Restriction endonucleases
molecular scissors – they cut DNA
restriction enzymes are highly specific. They cut DNA only within very
precise recognition sequences.
Pst 1
EcoR1
Sma1
Enzym
e
Organism from which
derived
Target
sequence
(cut at *)
5' -->3'
Ava I
Anabaena variabilis
C* C/T C G A/G
G
Bam HI
Bacillus amyloliquefaciens
G* G A T C C
Bgl II
Bacillus globigii
A* G A T C T
Eco RI
Escherichia coli RY 13
G* A A T T C
Eco RII
Escherichia coli R245
* C C A/T G G
Hae III
Haemophilus aegyptius
GG*CC
Hha I
Haemophilus haemolyticus
GCG*C
Hind III
Haemophilus inflenzae Rd
A* A G C T T
Hpa I
Haemophilus parainflenzae
G T T * AA C
Kpn I
Klebsiella pneumoniae
G GTAC * C
Sma I
Serratia marcescens
CCC*GGG
Sal I
Streptomyces albus G
G*TCGAC
Xma I
Xanthamonas malvacearum
C*CCGGG
DNA ligase
• Enzyme
• is molecular glue- sticks DNA together
• H bonds are not enough to hold sticky ends together. A means of
reforming the internucleotide linkage between 3’OH and
5’phosphate groups is required and ligase does this