Download The amount of DNA, # of genes and DNA per gene in various

Chapter 2
DNA Structure
and DNA
Manipulation
Jones and Bartlett Publishers © 2005
The amount of DNA, number of genes
and DNA per gene in various organisms
Organism
Genome size
# of genes
DNA/gene
•
Haemophilus influenzae
1.8 Mb
~1,700
~ 1 Kb
•
Escherichia coli
4.6 Mb
~4,300
~ 1 Kb
•
Baker’s Yeast
(Saccharomyces cerevisiae) 12.1 Mb
~6,000
~ 2 Kb
97 Mb
~18,000
~5.4 Kb
(Drosophila melanogaster)
185 Mb
~14,000
~13 Kb
•
Human (Homo sapiens)
3,000 Mb
~35,000
~ 86 Kb
•
A flowering plant
100 Mb
~25,000
~ 4 Kb
•
A worm
•
(Caenorhabditis elegans)
•
Fruit fly
(Arabidopsis thaliana)
DNA fragments can be cut and amplified
(cloned) in a bacterial cell
Structure of the 4 bases found in DNA
Base + sugar is called a nucleoside
Base + sugar + phosphate is called a nucleotide
Structure of a DNA (polynucleotide) chain
Polynucleotide
chains
have polarity.
One end has
5’-phosphate
and the other end
has 3’-OH
Structure of DNA Double Helix
Ribbon and space-filling diagrams
DNA has grooves of 2 sizes
Structure of A-T and G-C base pairs
Hydrogenbonds
are shown
as dotted
red lines.
A-T base
pairs have 2
and
G-C base
pairs have 3
H-bonds
H-bonds
are shown
as thin
flat white
disks in the
center
DNA strands are anti-parallel
5’
3’
3’
5’
The first proof was provided
In 1961 by measuring the
ratio of different dinucleotides
in DNA. The concentration of
5’AG3’ was equal to 5’CT3’ (as
expected from an antiparallel
orientation) and not equal to
5’TC3’ (as expected from a
a parallel orientation).
DNA sequencing in 1970s
confirmed this conclusion.
Restriction enzymes cleave DNA at a
specific sequence
Properties of restriction enzymes-1
(The next slide shows actual recognition sequences & cuts)
• DNA recognition sequence is usually 4-8 bp.
• The recognition sequence is usually a palindrome.
• The recognition sequence may be ambiguous (for example,
PuGCGCPy or CCTNAGG).
• The enzymes are named after the organisms from which they
were isolated.
• The cuts may result in blunt or sticky-ends.
• The sticky-ends may have 5’- (EcoRI, for example)or 3’overhangs (PstI, for example).
• The average distance between cutting sites is determined by
how long the recognition sequence is and the probability of
finding each nucleotide.
Properties of restriction enzymes-2
HaeIII
Haemophilus aegiptius
GG/CC
Blunt cut
Sau3A
Staphylococcus aureus
/GATC
5’-overhang
HhaI
Haemophilus haemolyticus
GCG/C
3’-overhang
SmaI
Serratia marcescens
CCC / GGG
Blunt cut
EcoRI
Escherichia coli RY13
G / AATTC
5’-overhang
PstI
Providencia Stuartii
CTGCA / G
3’-overhang
HaeII
Haemophilus aegiptius
RGCGC / Y
Ambiguous
sequence
NotI
Nocardia otitidis
GC /
GGCCGC
8 nt sequence
Size separation of DNA fragments
by electrophoresis in agarose gels
DNA is negatively charged due to phosphates on its surface.
As a result, it moves towards the positive pole.
Distance migrated by a DNA fragment
in a gel is related to log10 of its size
A “Restriction Map” shows the relative
location of DNA fragments
(A) Arrangement of EcoRI fragments (1 to 6) in bacteriophage l DNA
(B) Arrangement of BamHI fragments (1 to 6) in l DNA
Heating of DNA leads to the separation
of the 2 strands
Single stranded (SS) DNA can pair with a
complementary strand to regenerate DS DNA
Southern Blotting
DNA fragments separated in a gel can be transferred to a membrane for
hybridization to a SS DNA Prob. The extent of hybridization can be
quantitated by using a radioactive DNA probe and auto-radiography
DNA synthesis is done by an enzyme (DNA polymerase)
adding nucleotides to the 3’-end of a primer DNA chain
Polymerase Chain Reaction (PCR)-1
A pre-defined DNA
sequence in the
genome can be greatly
amplified by repeated
Polymerization cycles
using 2 primers which
hybridize to the ends
of the target DNA. In
each cycle, the amount of
target DNA
is doubled. After 10, 20
and 30 cycles, there is a
1000-, million- and
billion-fold amplification
respectively.
Polymerase Chain Reaction (PCR)-2
Each PCR cycle
has 3 stepsa. Melting of
DNA
b. Hybridization
of primer
c. DNA synthesis
Some terms used in Genetics
Genotype- The genetic constitution of an organism.
Phenotype- The visible appearance of an organism.
Homologous chromosomes- in a diploid organism, the 2 copies
of a chromosome inherited from the mother and the father.
Locus- Location of a gene on a chromosome.
Allelomorph (allele)- different versions of the same gene.
Homozygous- the 2 copies of a gene are identical.
Heterozygous- the 2 copies of a gene are different.
Restriction site Polymorphism
a. A mutation in the recognition sequence of a restriction enzyme can
lead to the gain or loss of a cutting site in a DNA sequence.
b. A restriction sequence with more than one variant is called
polymorphic site.
Restriction Fragment Length
Polymorphism (RFLP)
Variation in restriction
nuclease cutting
sequence at a
particular site creates
DNA fragments
of different sizes.
In a homozygous
organism the
fragments
are of the same size.
In a heterozygous
organism, the
fragments are of
different sizes.
DNA polymorphisms based on differences in
fragment sizes after DNA amplification
• Randomly amplified polymorphic DNA
(RAPD)
• Amplified fragment length polymorphisms
(AFLP)
• Simple Tandem Repeat polymorphism
(STRP)
tandem repeat sequences can also be studied
using restriction nucleases
Randomly amplified polymorphic DNA
(RAPD)
PCR is done with a
single short primer that
hybridizes to many
places in a genome.
Occasionally, 2
primers hybridize to
complementary strands
near each other
generating a PCR
product. Different
organisms of a
species have slightly
different DNA sequences
which yield mostly the
same PCR products,
but a few PCR products
are gained or lost.
Amplified Fragment length Polymorphism
(AFLP)
DNA is cut with a
restriction enzyme
and an adapter DNA
containing primer
hybridization sequence
is hybridized and joined
(ligated) to the
sticky ends. PCR
amplification leads
to different size
products. Addition
of extra nucleotides
to the hybridization end
of the adapter reduces
the number of PCR
products generated.
Simple Tandem Repeat Polymorphism (STRP)
Variants (morphs)
with different number
of repeats of a short
sequence can be
distinguished
by amplification of
the entire repeated
target by PCR, using
2 primers that
hybridize in flanking
DNA. More repeats
lead to larger
PCR product.
Disease genes can be mapped using
DNA markers
Of the estimated 35-40,000 genes in the human genome, only ~8,000
genes are known. A current list can be found at
http://www3.ncbi.nlm.nih.gov/Omim/mimstats.html. In contrast,
many more DNA markers are known. Therefore, it is easier to map a
new gene using a DNA marker (see Chapter 5 for methods of gene mapping)