DNA: Structure, Replication, and Expression
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A. Structure of DNA
1. Discovered by James Watson and Francis
Crick in 1953
2. Stores genetic information for the
production of proteins
3. Composed of nucleotides
A) A nucleotide is composed of a
nitrogenous base, sugar, and phosphate
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1) The 4 bases of DNA are adenine, guanine,
thymine, and cytosine
2) Sugar – deoxyribose
3) Phosphate – PO
4
B) Bases attach to 1’ carbon
C) Hydroxyl (-OH) group on the 3’ carbon
D) Phosphate group on 5’ carbon
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4. Nucleotides arranged in chains (strands)
A) The ends of each strand are identified by
the number of the carbon nearest to that end
5. Bases are held together by H
+
bonds
A) Base Pairing Rules
1) A with T
2) G with C
6. DNA is a complementary, antiparallel, double
helix
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B. DNA Replication
1. Semi-Conservative Replication
A) Results in 2 molecules; each with 1
original strand and 1 new strand
B) Uses multiple enzymes:
1) DNA helicase – unwinds the double
helix
a) Breaks H
+
bonds between bases
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2) DNA Polymerase III – builds new strands
a) Cannot add nucleotides without one
already being present (cannot start from
scratch)
i) Requires an
RNA primer
(a) Small section of RNA that initiates
DNA replication
(i) Created by RNA primase (can
start from scratch)
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b) Can only add new nucleotides to the 3’
end of an already existing chain
i) Therefore the enzyme must read the
original in a 3’ to 5’ direction and the
new DNA is built in a 5’ to 3’ direction
3) DNA Polymerase I – removes & replaces
the RNA primer(s) with DNA
4) DNA ligase – joins together DNA fragments
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C. DNA Expression
1. Transcription-Translation
A) Process by which information is taken
from DNA and used to make proteins
2. Transcription
A) Process by which RNA is created
from a DNA template
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B) RNA polymerase “reads” the template DNA
strand and creates a complementary RNA
strand
1) It recognizes
promoter
and
termination
regions on the DNA template
3. Translation
A) Process by which a polypeptide strand is
created from a mRNA template
B) Occurs in the ribosome
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C) Ribosome begins “reading” the mRNA strand
until it reaches the start codon (AUG)
1) Codon – three-nucleotide sequence that
represents one amino acid
D) tRNA brings in the appropriate amino acid for
the AUG codon
E) The next codon is read and another amino
acid comes in forming a peptide bond with the
previous one
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F) This continues until the ribosomes reaches a
stop (nonsense) codon on the mRNA
1) Can be UAA, UAG, or UGA
G) The new protein is released to the cell for use
or release
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Genetic Engineering
A. Tools & Techniques
1. DNA strand properties
A) Strands denature (separate) at
near-boiling temperatures
B) Strands renature (reform) as the
strands cool
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2. Enzymes
A) Restriction endonucleases
1) Capable of recognizing specific
sequences and clipping the DNA at that
location
2) Used to splice pieces of DNA into
plasmids and chromosomes
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B) DNA Ligase
1) Used to rejoin DNA fragments
C) DNA & RNA polymerases
1) Used to make new strands of DNA or
RNA (respectively)
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D) Reverse transcriptase
1) Uses an RNA template to create a
strand of DNA
2) Can be used to help sequence RNA or
examine DNA with the “junk” segments
removed
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3. Visualization
A) Electrophoresis
1) Uses an electrical current and a
gelatin-like medium to create a visual
pattern of DNA fragments
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4. Oligonucleotides
A) Smaller segments of DNA created for
research purposes
B) Most eukaryotic DNA is too large to
study as a whole; oligonucleotides allow it
to be studied in pieces and then
information is consolidated to observe the
whole genome
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C) Oligonucleotides can also be sequenced
using a sequencer
1) Can sequence 500-10 million bp
segments at a rate of about 1000 bp/day
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5. Polymerase Chain Reaction (PCR)
5. Polymerase Chain Reaction (PCR)
A) Allows one to create millions of copies of a
A) Allows one to create millions of copies of a
specific segment of DNA
specific segment of DNA
B) Most often know in advance the segments
B) Most often know in advance the segments
to be copied
to be copied
C) Requires:
C) Requires:
1) A sample of the original DNA with the
segment to be copied
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2) The appropriate endonucleases to extract the
desired segment
3) The appropriate RNA primer to initiate
replication of that segment
4) DNA polymerase III to make copies of that
segment
5) A thermal cycler to heat and cool the DNA so it
can denature and renature multiple times
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B. Recombinant DNA
1. Recombination
A) The transfer of DNA from one organism
to another
1) Results in a new genetic strain
different from the donor and original
recipient
2) Normally involves the transfer of
plasmids
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3) The recipient organism must accept the
DNA into its genetic make-up, express it,
and pass it on to its offspring for the transfer
to be successful
4) Can occur naturally or artificially
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B) 3 mechanisms of natural transfer
1) Conjugation
a) Involves a pilus
b) Pilus forms a bridge between the 2
organisms and a replicated plasmid
is transferred
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c) Only certain bacteria are able to produce
a pilus but that trait is also transferred
through this process
d) Generally limited to organisms of the
same genus
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2) Transformation
a) A cell “picks up” a piece of DNA from
the environment usually from a lysed
cell
b) Allows a cell to get genetic material
from an entirely different species
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3) Transduction
a) Involves infection by bacteriophage
b) The phage carries genetic material
from its previous host cell into the new
host
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C) Genes can also be introduced artificially
1) Transfection – process of artificially
introducing genes into organisms
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a) Transfected orgs. may be patented
b) Can be microbes, plants, or animals
i) Bacteria
(a) Plays a major role in agriculture
(i) Can act as insecticides
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ii) Viruses
(a) Often used to transmit the gene into
another organism
(i) Vector – a microbe or virus that transmits
material (wanted or unwanted) into a host
organism
(b) Gene therapy, experimental vaccines, and
pathogenic testing all use transfected
viruses
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iii) Plants
(a) Can introduce a number of new
characteristics
(i) Herbicide resistance
(ii) Built-in insecticide
(iii) Microbial resistance
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iv) Animals
(a) Serve many valuable purposes
(i) Production of human hormones,
antibodies, and enzymes
(ii) The study of human disorders
(iii) Healthier meat products
(iv) Replacement tissues and organs
(v) Nearly all early research involving
human genetic therapy
DNA, discovered by Watson and Crick, stores genetic information in nucleotides consisting of nitrogenous bases, sugar, and phosphate. It forms a double helix with adenine, guanine, thymine, and cytosine bases following specific pairing rules. DNA replication is semi-conservative using enzymes like helicase, DNA polymerase III, RNA primase, and ligase. Expression involves transcription and translation to produce proteins.
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Presentation Transcript
Microbial Genetics Microbial Genetics A. Structure of DNA 1. Discovered by James Watson and Francis Crick in 1953 2. Stores genetic information for the production of proteins 3. Composed of nucleotides A) A nucleotide is composed of a nitrogenous base, sugar, and phosphate
Microbial Genetics Microbial Genetics 1) The 4 bases of DNA are adenine, guanine, thymine, and cytosine 2) Sugar deoxyribose 3) Phosphate PO4 B) Bases attach to 1 carbon C) Hydroxyl (-OH) group on the 3 carbon D) Phosphate group on 5 carbon
Microbial Genetics Microbial Genetics 4. Nucleotides arranged in chains (strands) A) The ends of each strand are identified by the number of the carbon nearest to that end 5. Bases are held together by H+bonds A) Base Pairing Rules 1) A with T 2) G with C 6. DNA is a complementary, antiparallel, double helix
Microbial Genetics Microbial Genetics B. DNA Replication 1. Semi-Conservative Replication A) Results in 2 molecules; each with 1 original strand and 1 new strand B) Uses multiple enzymes: 1) DNA helicase unwinds the double helix a) Breaks H+bonds between bases
Microbial Genetics Microbial Genetics 2) DNA Polymerase III builds new strands a) Cannot add nucleotides without one already being present (cannot start from scratch) i) Requires an RNA primer (a) Small section of RNA that initiates DNA replication (i) Created by RNA primase (can start from scratch)
Microbial Genetics Microbial Genetics b) Can only add new nucleotides to the 3 end of an already existing chain i) Therefore the enzyme must read the original in a 3 to 5 direction and the new DNA is built in a 5 to 3 direction 3) DNA Polymerase I removes & replaces the RNA primer(s) with DNA 4) DNA ligase joins together DNA fragments
Microbial Genetics Microbial Genetics C. DNA Expression 1. Transcription-Translation A) Process by which information is taken from DNA and used to make proteins 2. Transcription A) Process by which RNA is created from a DNA template
Microbial Genetics Microbial Genetics B) RNA polymerase reads the template DNA strand and creates a complementary RNA strand 1) It recognizes promoter and termination regions on the DNA template 3. Translation A) Process by which a polypeptide strand is created from a mRNA template B) Occurs in the ribosome
Microbial Genetics Microbial Genetics C) Ribosome begins reading the mRNA strand until it reaches the start codon (AUG) 1) Codon three-nucleotide sequence that represents one amino acid D) tRNA brings in the appropriate amino acid for the AUG codon E) The next codon is read and another amino acid comes in forming a peptide bond with the previous one
Microbial Genetics Microbial Genetics F) This continues until the ribosomes reaches a stop (nonsense) codon on the mRNA 1) Can be UAA, UAG, or UGA G) The new protein is released to the cell for use or release
Microbial Genetics Microbial Genetics Genetic Engineering A. Tools & Techniques 1. DNA strand properties A) Strands denature (separate) at near-boiling temperatures B) Strands renature (reform) as the strands cool
Microbial Genetics Microbial Genetics 2. Enzymes A) Restriction endonucleases 1) Capable of recognizing specific sequences and clipping the DNA at that location 2) Used to splice pieces of DNA into plasmids and chromosomes
Microbial Genetics Microbial Genetics B) DNA Ligase 1) Used to rejoin DNA fragments C) DNA & RNA polymerases 1) Used to make new strands of DNA or RNA (respectively)
Microbial Genetics Microbial Genetics D) Reverse transcriptase 1) Uses an RNA template to create a strand of DNA 2) Can be used to help sequence RNA or examine DNA with the junk segments removed
Microbial Genetics Microbial Genetics 3. Visualization A) Electrophoresis 1) Uses an electrical current and a gelatin-like medium to create a visual pattern of DNA fragments
Microbial Genetics Microbial Genetics 4. Oligonucleotides A) Smaller segments of DNA created for research purposes B) Most eukaryotic DNA is too large to study as a whole; oligonucleotides allow it to be studied in pieces and then information is consolidated to observe the whole genome
Microbial Genetics Microbial Genetics C) Oligonucleotides can also be sequenced using a sequencer 1) Can sequence 500-10 million bp segments at a rate of about 1000 bp/day
Microbial Genetics Microbial Genetics 5. Polymerase Chain Reaction (PCR) A) Allows one to create millions of copies of a specific segment of DNA B) Most often know in advance the segments to be copied C) Requires: 1) A sample of the original DNA with the segment to be copied
Microbial Genetics Microbial Genetics 2) The appropriate endonucleases to extract the desired segment 3) The appropriate RNA primer to initiate replication of that segment 4) DNA polymerase III to make copies of that segment 5) A thermal cycler to heat and cool the DNA so it can denature and renature multiple times
Microbial Genetics Microbial Genetics B. Recombinant DNA 1. Recombination A) The transfer of DNA from one organism to another 1) Results in a new genetic strain different from the donor and original recipient 2) Normally involves the transfer of plasmids
Microbial Genetics Microbial Genetics 3) The recipient organism must accept the DNA into its genetic make-up, express it, and pass it on to its offspring for the transfer to be successful 4) Can occur naturally or artificially
Microbial Genetics Microbial Genetics B) 3 mechanisms of natural transfer 1) Conjugation a) Involves a pilus b) Pilus forms a bridge between the 2 organisms and a replicated plasmid is transferred
Microbial Genetics Microbial Genetics c) Only certain bacteria are able to produce a pilus but that trait is also transferred through this process d) Generally limited to organisms of the same genus
Microbial Genetics Microbial Genetics 2) Transformation a) A cell picks up a piece of DNA from the environment usually from a lysed cell b) Allows a cell to get genetic material from an entirely different species
Microbial Genetics Microbial Genetics 3) Transduction a) Involves infection by bacteriophage b) The phage carries genetic material from its previous host cell into the new host
Microbial Genetics Microbial Genetics C) Genes can also be introduced artificially 1) Transfection process of artificially introducing genes into organisms
Microbial Genetics Microbial Genetics a) Transfected orgs. may be patented b) Can be microbes, plants, or animals i) Bacteria (a) Plays a major role in agriculture (i) Can act as insecticides
Microbial Genetics Microbial Genetics ii) Viruses (a) Often used to transmit the gene into another organism (i) Vector a microbe or virus that transmits material (wanted or unwanted) into a host organism (b) Gene therapy, experimental vaccines, and pathogenic testing all use transfected viruses
Microbial Genetics Microbial Genetics iii) Plants (a) Can introduce a number of new characteristics (i) Herbicide resistance (ii) Built-in insecticide (iii) Microbial resistance
Microbial Genetics Microbial Genetics iv) Animals (a) Serve many valuable purposes (i) Production of human hormones, antibodies, and enzymes (ii) The study of human disorders (iii) Healthier meat products (iv) Replacement tissues and organs (v) Nearly all early research involving human genetic therapy
