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Biotechnology Research
BIT 220
MCCC
Chapter 10
Terminology
Molecular Biology/Genetics: the study of gene structure
and function at the molecular level
Molecular Biotechnology: the ability to transfer
specific units of genetic information from one organism
to another
Recombinant DNA Technology
= gene splicing
= genetic engineering
=gene transplantation
= gene cloning
Genetics
• Science of Heredity
• Explains similarities and differences between organisms
• Two Branches
– Classical (Mendelian)
• cells contain pairs of ‘factors’ which determine physical
characteristics
• these factors segregate during meiosis, independently
– Molecular
DNA/RNA Molecular Basis of
Life
Central Dogma
DNA
RNA
control
Transcription
protein
DNA - Deoxyribonucleic Acid
RNA - Ribonucleic Acid
Translation
DNA
DNA
Replication (mitosis,meiosis)
DNA Structure
Replication
Gene Expression
Mutation
PROPERTIES OF DNA
1.Replicate
Genotypic function
2.Mutate –
chemically change and transmit these changes to future
generations - if mutant in germ line
evolutionary function
3.Gene expression
Direct the synthesis of proteins
phenotypic function
1. Miescher DNA
1868 isolated DNA from pus cells (NUCLEIN)
2. Frederic Griffith 10.1 and 10.2
1928 ‘transforming principle’ in pneumococcus
3. Oswald Avery, Colin MacLeod, Maclyn McCarty 10.3
1944 DNA was the active substance in the
heat-killed S strain extracts because DNases
destroyed the activity while RNases and proteases did no.
4. Chargaff
the molar concentration of A = T and G = C
which led to the discovery of base pair
complementarity.
5. Wilkins and Franklin 10.10
X-ray crystallographs (diffraction patterns) of
DNA -Review DNA structure: Figures 10.6, 10.7,
10.8, 10.11, 10.12, 10.13, Table 10.3
6. Watson and Crick
deduced a two-stranded structure
wrapped in a right-handed helix with the bases
internal,
the phosphates external, and an internal
repeating subunit
separated by 0.34 nm
Nucleotide
Smallest unit of nucleic acid
Three components
1. sugar
2. phosphate
3. nitrogenous base
Other Nomenclature
Nucleoside : SUGAR and BASE (NO PHOSPHATE)
BASES
NUCLEOSIDES
NUCLEOTIDES
Adenine
Guanine
Cytosine
Thymine
Deoxyadenosine
Deoxyguanosine
Deoxycytidine
Deoxythymidine
Deoxyadenosine 5’-triphosphate
“”
(dATP)
“”
“”
Double Helix
2 strands wrapped around
one another
SPIRAL STAIRCASE
Allows replication
Allows permanence
Bases on one side of helix are
complementary to other strand
What draws two strands together?
1. Bases hydrogen bond
2. Bases are hydrophobic
DNA Structure
A. Sugar-Phosphate Backbone
covalent bonds between S and P
phosphodiester linkages
B. Stairs
Sugar covalently bound to base
Bases hydrogen bound to each other
A::T
C:::G
DNA Antiparallel Strands
One strand runs 3’C (OH of
sugar) to 5’ C (phosphate);
The other strand 5’ to 3’
Alternate forms
A 11 bp per turn/ right-handed
B 10.4 base pairs per turn/ right handed FOUND IN VIVO physiological DNA form
Z 12 bp per turn/left handed
Supercoiling
In vivo negatively supercoiled (underwound)
Introduced when one or both strands nicked
and strands rotate around one another
Figures 10.14 and 10.15
Chromosome Structure
Composed of DNA (RNA viruses) and proteins
single piece of nucleic acid
Prokaryotic (bacteria and virus)
single chromosome/ 3000 genes
E coli condenses DNA into loops 10.16
no introns
all single copy DNA
monoploid
Eukaryotic
1000 times more DNA than bacteria
noncoding regions
introns
30-85% single copy
most diploid
Eukaryotic Chromosomes
Chromatin
a. DNA
b. non-histone proteins
c. histone proteins: 10.23 and 10.24
DNA wraps around dimers of each
H2a, H2b, H3, and H4 forming nucleosome
H1 sits outside nucleosome
DNA Compaction
10.27
Level 1 Nucleosome
Level 2 supercoiling of nucleosomes
Level 3 Scaffold composed of non-histone proteins
packing during metaphase (most condensation)
Centromeres Figures 10.29 and 10.30
interchangeable among chromosomes
110-120 bp in length.
Telomeres Figure 10.31
contain special repeated DNA sequences that enable
the ends of the chromosomes to be replicated, inhibit their
degradation by DNA degrading enzymes, and prevent fusion
with other chromosomes.
In vertebrates, the TTAGGG repeat is highly conserved and in
humans 500-3000 repeats occur in telomeres which gradually
shorten with age.
Eukaryotic DNA contains excess DNA, up to 50% or more, that does
not code for proteins and comprises families of highly repeated sequence
elements or repetitive DNA (satellite DNA)
three classes of DNA
1. unique or single-copy
1-10 copies per haploid genome
genes that encode proteins
regulatory sequences
2. moderately repetitive
10-100,000 copies
transposable elements (jumping genes)
rRNA
histone
ribosomal genes
3. highly repetitive
>100,000 copies
telomeres
centromeres
unknown function
Types of repeats
LINEs - Long Interspersed Nuclear Elements
SINEs - Short Interspersed Nuclear Elements
LTRs - Long terminal repeats
DNA transposons (transposable elements)
Denaturation/Renaturation
D: Two strands of DNA separate (Melt)
R: Two strand hydrogen bond through complementary bases
(Annealing)
The kinetics of these processes will indicate the amount of
repetitive DNA
Higher the copy number; the faster the renaturation occurs
Hybridization
Radioactive-labelled sequences
Nucleic Acid probe contains radioactive isotope
this isotope emits charged particles as it decays
these charged particles captured on emulsion (film)
exposed silver halides produce back spots
Fluorecently-labelled sequences
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