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Unit 3 Molecular Genetics

Grade 12 Biology (Columbia College (Canada))

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Chapter 5: The Structure and Function of DNA

Section 5.1: DNA Structure and Organization in the Cell

Identifying DNA as the Material of Hereditary:

-1928, London England, Frederick Griffith (Microbiologist) was studying bacterial pneumonia
specifically Streptococcus pneumoniae
-Two forms of the bacteria were used
o S-strain: highly pathogenic but could be made non-pathogenic by heating it
o R-strain: non-pathogenic form
-Discovered that mice died after being injected with a mixture of heat-killed S strain and living
R-strain
-Concluded that the S-strain had passed on its deadly properties to the R strain.
-Called this the transforming principle in a scientific paper published in 1928.
-Died of injuries during WWII but work resulted in further research.
-Oswald Avery (Canadian-American Microbiologist) was studying the same bacteria and after
Griffith’s paper, focused his work on identifying the molecules that caused this
transformation.

-1944: Oswald Avery, Colin MacLeod, Maclyn McCarty published study that supported the
hypothesis that DNA was the hereditary material.
• Added enzymes to extracts of heat-killed S-strain that they mixed with live R-strain.
Only one that did not cause transformation of the R-strain to the pathogenic S-strain
was the enzyme that had the DNA-destroying enzyme (others had destroyed protein,
and destroyed RNA)
• DNA was the transforming principle in Griffith’s research.

1952: Alfred Hershey and Martha Chase (American microbiologists) ruled out protein in
favour of DNA as the hereditary material (still believed at the time and this was one of the
most famous experiments to do this)
• Used bacteriophages which are viruses that infect bacteria, have a inner nucleic acid
core and outer protein coat called a capsid.
• Aimed to determine which part of the virus- the DNA in the viral core or the protein
the capsid enters bacterial cells and directs the production of more viruses.
• Used T2 bacteriophage which has a protein coat surrounding a piece of DNA which will
attach to the outside of the bacterium and inject its genetic information into it.
• Used radioactive isotopes to trace each type of molecule by adding a radioactive
source of sulfur into the protein of the virus (as DNA does not have sulfur) by growing
infected bacteria in media that contain the isotope. Since DNA contains phosphorous,
they were able to produce a virus that had DNA labelled with a radioactive isotope of
P.

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Experiments
Experiment 1: Virus with radioactive DNA infected an E.coli bacteria and it was found that the
radioactive DNA was in the bacteria and not the liquid medium.

Experiment 2: Virus with radioactive protein infected an E.coli bacteria and the radioactive
protein was found in the liquid medium and not the bacteria.

This proved that the viral DNA was transferred to bacterial cells and that the viral DNA held
the genetic information needed for viruses to reproduce.

Determining the Chemical Composition and Structure of DNA

1869- Friedrich Miescher isolated the nuclei of white blood cells obtained from pus soiled
hospital bandages. Extracted a weakly acidic substance containing nitrogen and phosphorous.
Named it nuclein (as it was found in the nuclei but later would be renamed nucleic acid).

1900s- Phoebus Levene (Russian-American biochemist) isolated two types of nucleic acid. He
named one ribose based on the 5-carbon sugar called ribose. This is now called RNA or
ribonucleic acid.
Other one had a similar 5-carbon sugar but with one less oxygen atom. He named it
deoxyribose nucleic acid, later renamed deoxyribonucleic acid or DNA.

1919- Levene proposed that RNA and DNA were made of nucleotides- repeating unit of
nucleic acids, composed of a sugar group, phosphate group and a nitrogenous base; after
analysing the results of several hydrolysis reactions of nucleic acid from yeast.
-Proposed that nucleic acids were made of long chains of nucleotides called the
polynucleotide model of nucleic acids.
-Incorrectly hypothesized that nucleic acids had a tetranucleotide structure in which 4 were
linked together in a repeating sequence (very simple model) and helped to continue the belief
that protein was the material of hereditary.

DNA structure
- DNA and RNA made of combination of
4 nucleotides.
- Each composed of 5-C deoxyribose
sugar, phosphate group, nitrogen
containing base
- Linked together by covalent bonds
- Purine bases: Adenine (A) and Guanine
(G)- Two fused rings
- Pyrimidines: Cytosine (C ) and Thymine
(T) – Single ring
- RNA has Uracil (U) as a pyrimidine
(instead of T)

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-1940s, Erwin Chargaff (Austrian-American Biochemist) started research to study the

chemistry of Nucleic Acids after reading Avery’s work.
Two important conclusions:
- Showed there is variation in the composition of nucleotides among different species
(disproved Levene)
- Demonstrated that all DNA keeps certain properties even if its composition changes.
- Observed that the nucleotides were always present in certain proportions. Example:
Amount of adenine in a sample is always about equal to the amount of thymine, and
that C = G.

Chargaff’s Rule: in DNA, the percent composition of A =T, and the % composition of C= G.

1950s- James Watson (American Biologist) and Watson Crick (British Physicist) at the
university of Cambridge decided to determine the structure of DNA using data already
collected particularly from:

1951- Linus Pauling (American Chemist) discovered that many proteins have helix-shaped
structures using his method of assembling 3D models based on known distances and bond
angles between atoms in molecules.

1950s- Rosalind Franklin (British Chemist) with Maurice Wilkins were using X-ray diffraction to
analyze biological molecules in which X-rays are bent (diffracted) as they encounter molecules
and this pattern can be captured on film and used to infer structure.
She concluded that DNA had a helical structure with two regularly repeating patterns.
Also based on how DNA reacted with water, she concluded that the bases were on the inside
of the structure and the sugar-phosphate backbone was on the outside (toward the watery
nucleus of the cell).

Watson and Crick built a model of DNA using cardboard cut-outs to represent components of
nucleotides. They deduced that DNA has:
- Twisted, ladder-like structure called a double helix
- Sugar-phosphate molecules make up the sides and the bases make up the rungs
- Knew from Rosalind that the distance between the sugar-phosphate molecules
remained constant over the length of the molecule
- Chargaff’s rule helped them infer that an A on one chain is always across from a T, C
across from a G
- Base pairs can be in any order (allows for differences in structure between species)
1953- published a two-page paper of their model

Modern DNA Model:

- 2 polynucleotide strands that twist around each other = double helix
- Each has a backbone of alternating phosphate groups and sugars
- Bases are attached to each sugar and protrude inward at regular intervals
- Constant total distance between the sugar-phosphate backbone

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- 2 strands of DNA are complementary to each other. Purine always pairs with a
pyrimidine: A-T and G-C.
- Complementary Base-Pairing: interaction of bases of nucleotides on opposite strands
through the formation of hydrogen bonds

- Hydrogen bonds link each complementary base pair. A/T share 2 bonds. G/C share 3.
- 2 strands of DNA are antiparallel- the directionality of the 2 strands in a DNA
molecule; the strands run in opposite directions with each end of a DNA molecule
containing the 3’ end of one strand and the 5’ end of the other. This comes from the
numbering of the carbons on the deoxyribose sugar (Phosphate group is on the 5’
carbon, the OH is on the 3’ carbon)

- Sequence of DNA is always written in the 5’ to 3’ direction.

- Ribbon model of DNA: shows the components of DNA. Space-

filling model shows the surface of DNA (sugar-phosphate on the
outer, and the bases in the inner part)

Structure and Organization of Genetic Material in Prokaryotes and

Eukaryotes

Genome: the complete genetic makeup of an organism; an organism’s total DNA sequence

Gene: the basic unit of hereditary that determines, in whole or part, a genetic trait; a specific
sequence of DNA that encodes for proteins and RNA molecules, and can contain sequences
that influence production of these molecules

-large amount of the DNA in the genome of an organism does not contain instructions for
producing proteins and is non-coding.

DNA in Prokaryotic Cells:

-DNA in the form of a circular, double-stranded molecule
• can have more than one copy of a chromosome
• do not have nuclear membrane so chromosome is packed tightly in a specific region of
the cell
-Nucleoid: structure in bacteria that contains the chromosomal DNA

-compacting of bacterial chromosomal DNA is

1000x
-specialized proteins bind to the DNA and help
fold sections o the chromosomes into loop-like
structures. This compacts it 10x more.

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DNA supercooling: the formation of additional coils in the structure of DNA due to twisting
forces on the molecule
-twists cause changes in the conformation that result in a section of DNA coiling in onto itself.
-amount of supercooling is controlled by two enzymes: topoisomerase I and topoisomerase II
(essential for survival).
-antibacterial drugs have been developed to target and block the activity of T II. Ex.
Quinolones and coumarin drugs.

Plasmids: circular or linear DNA molecules that are

not part of the nucleoid and carry non-essential
genes. Can be present in prokaryotes having one or
more.
Can be copied or transmitted between cells or added
into the cell’s chromosomal DNA and are reproduced during cell division (can be transferred
from cell to cell).

Most are haploid with only one copy of each gene.

Genomes contain very little non-essential DNA. Most have regions of genes or regulatory
sequences.
Regulatory Sequence: a sequence of DNA that regulates the activity of a gene. (determine
when certain genes and the associated cell function are activated)

DNA of Eukaryotic Cells:

-much greater total DNA than Prokaryotes

-genetic material is within the nucleus
-Mitochondria and chloroplasts contain some cellular DNA
-much greater compacting of DNA through different levels
of organization.

1) Simplest level: DNA associates with family of

proteins: histones (a member of a family of
proteins that associate with DNA in eukaryotic
cells which acts to help compact the DNA) to form
repeated series of structures called nucleosomes.
Nucleosomes: condensed structure formed when double
stranded DNA (146 base pairs in length) wraps around an
octamer of histone proteins. Connected by regions called
linker DNA.
2) Further compacting occurs by the coiling of
nucleosomes with the aid of H1 histone proteins.
(DNA has been compacted 50 times)
3) Formation of radial loop domains of the 30nm fibre
which are anchored to a supporting scaffold of proteins in the nucleus.

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4) 30 nm fibre of looped domains called euchromatin undergoes further compacting into

heterochromatin. Chromatin will condense and become visible as distinct
chromosomes during cellular division.
Chromatin: non-condensed form of genetic material that predominates for most of the
eukaryotic cell cycle; consists of a complex of DNA and proteins.

Most eukaryotes are diploid: contain two copies of each gene.

Some (ferns and algae) are Haploid -contain one copy of each gene.
Bred organisms (seedless watermelon) are triploid

-genes are not evenly spaced along a chromosome and not equally divided among the
chromosomes
-size and number of genes in the eukaryotic genome vary a lot
-No correlation between organism complexity and genome size or number of protein-
coding genes (lack of: Humans have same number of protein-coding genes as a worm like
organism but 30 x more DNA)

-Genes vary in the molecules they produce. Broader than just a protein-coding sequence
as some can codes for RNA molecules that are needed for other cellular processes and do
not just form a protein directly.

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Section 5.2: DNA Replication

Production of new cells (rbc/skin cells, cells to repair damage) is achieved through mitosis
and cytokinesis.

Cell Cycle: lifecycle of a cell consisting of growth

stage (interphase), mitosis and cytoplasmic
division to produce two new cells.
Each new cell or daughter cell contains the same
genetic information as the original plant cell so
that it can carry on the same functions.
Each new cell requires an exact copy of the
parental DNA.
During S phase of Interphase, a cell copies all of
its DNA (happens once).

DNA Replication: the process of producing two

identical DNA molecules, from an original,
parent DNA molecule.

1953- Watson and Crick in their paper basically said that due to the complementary base
pairing each strand of DNA can serve as a template for the production of a new
complementary strand.
1 month later- 2nd paper published a paper on DNA replication proposing that the two
strands of DNA unwound and separated, and each nucleotide chain served as a template
for the formation of a new chain.

Three Proposed Models for DNA Replication:

1. Conservative Model: formation of two new
daughter strands from the parent templates and the
two new strands joined to create a new double helix.
The two original strands would reform into the
parent molecule.
2. Semi-conservative Model: Each new DNA
molecule would conserve half of the strand of the
original molecule (One proposed by Watson and
Crick).
3. Dispersive Model: Parental DNA molecules were
broken to fragments and both strands in each of the daughter molecules were made
up of an assortment of parents and new DNA.

Meselson and Stahl:

-Proposed models could be tested if they could distinguish between the original
parental strand and the newly synthesized daughter strand when DNA is copied.

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-Used 2 different isotopes of nitrogen (14N and 15N) as it is an essential DNA

component (brought into any new daughter cells) and the presence of a light/heavy
form of N meant that they could separate the DNA strands by how much of each
isotope there was using densities and centrifugation.

Experiment:
1. Bacteria were grown in a liquid culture medium of 15N
2. Grown until the population of bacterial cells only had this isotope in their DNA.
3. Transferred to a culture medium with only 14N.
4. Samples were removed just before transformation and as the bacteria divided.
5. DNA was isolated and samples were analyzed by centrifugation.

Findings:
1. DNA samples just prior to transfer were uniform
density and only had bands for 15N.
2. 1st round of replication: DNA with a single band
half way between 15N and 14N. Inferred the DNA
had one of each strand. Ruled out conservative
model because there would have been 2 bands of
each.
3. 2nd round of replication: DNA strand separated into
two distinct bands with one band being 14N only
DNA and the other to DNA that had one strand
with 14N and one with 15N.
4. Multiple rounds of replication- same two bands.
Ruled out Dispersive (one band observed).
Semi-conservative model was accepted and have
been supported by other studies.

DNA Replication:
Semi-conservation Replication: the mechanism of DNA replication in which each newly
synthesized DNA molecule is composed of one strand from the original DNA (parent DNA)
molecule and one new strand (daughter DNA).
Each resulting DNA molecule conserves half of the original molecule.

Overall:
1. Initiation: portion of DNA double helix is unwound to expose the bases for new pairing
2. Elongation Phase: 2 new strands of DNA are assembled using the parent DNA has a
template. New DNA molecules- each composed of one strand of parent DNA and one
daughter DNA re-form into double helices.
3. Termination: Replication is completed, and 2 new DNA molecules separate from one
another.

Process is described as in prokaryotes (E.coli) due to research studies. Events are taking place
simultaneously on the same DNA molecule.

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Initiation:
Origin of Replication: the DNA sequence (specific nucleotide sequence) where replication
begins.
Several initiator proteins bind to the DNA and begin to unwind the double helix.
Helicase: a group of enzymes that aid in unwinding the DNA. Cleave the hydrogen bonds that
link the complementary base pairs between strands together.
• Single strand-binding proteins stabilize the newly unwound single strands.
• Tendency if not checked to reform as a double helix.
• Single strand regions serve as the templates used to guide the synthesis of new
polynucleotide strands
• Topoisomerase II enzyme helps to relieve the strain on the double helix ahead of the
replication forks.
• Creates an unwound, oval-shaped area called a replication bubble with two Y shaped
regions at each end of the unwound area.
• Replication fork: each y shaped area.
• Each replication fork will move long the DNA in opposite directions

Elongation: synthesizes new DNA strands by joining individual nucleotides together.

DNA polymerase III enzyme catalyzes the addition of new nucleotides one at a time, creating
a strand of DNA that is complementary to the parental strand.
Only attaches new nucleotides to the free 3’ OH end of a pre-existing strand of nucleotides.
Can only synthesize a new strand from the parent strand in the 5’ to 3’ direction towards the
fork.

Separated double stranded DNA do not have free 3’OH end for DNA polymerase to begin.
DNA strands MUST be started with a short fragment of nucleotide sequences complementary
to the template.
For the leading strand this only needs to
happen once. DNA polymerase will keep
adding new nucleotides to the 3’ end as it
moves along the same direction as the
replication fork.
Synthesis of the other strand requires DNA
polymerase to move in the opposite direction
to the replication fork.
Lagging strand occurs in short fragments and
in a discontinuous manner.
Okazaki fragments: short DNA fragments that
are generated during the synthesis of the lagging strand in DNA replication.

Primers: in DNA replication, short segment of RNA that is complementary to a part of the 3’
to 5’ DNA template strand and serves as a starting point for addition of nucleotides.
(short nucleotide sequences use to start DNA replication).

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Synthesized by the enzyme primase

Primer is in place and DNA polymerase extends the strand adding new nucleotides to the free
3’ OH end.

Synthesis of the lagging strand (movement away from the replication fork) requires several
primers to be used.
Each primer is added, a new DNA
fragment is generated. Result is synthesis
of Okazaki fragments- short DNA
fragments that begin with a section of
DNA.
DNA Polymerase I: enzyme that removes
RNA primer and fills gaps between the
okazaki fragments on the laggings strand
with DNA nucleotides and proofreads
newly synthesized DNA.
-enzyme fills in the space by extending
the DNA fragments.
DNA Ligase: enzyme that joins the okazaki fragments.
Termination:
-only a short region of DNA is found in single strand form as replication fork progresses along
replicating DNA.
Newly formed strands rewind right away into chemically stable double helix.
Replication machine: protein-DNA complex at each replication fork that carries out
replication.
-two new molecules separate.

Correcting Errors:
Errors could include:
1) Mispairing between a new nucleotide and a nucleotide on the template strand.
Example: T paired with a G instead of A.
2) Strand Slippage causes additions or omissions of nucleotides due to newly
synthesizing strand lopping out allowing the addition of an extra nucleotide or the
looping out of the template strand and the nucleotide is omitted.
Fixing them:
1) DNA polymerase I and DNA Polymerase II: an enzyme that proofreads newly
synthesized DNA has a proofreading function. After each nucleotide is added to the
new DNA strand, they can recognize whether the nucleotide is the correct one.
Replication is stalled if an incorrect nucleotide is added as the 3’ hydroxyl end is in the
wrong position for the next nucleotide to bond. The DNA polymerases will cut out the
incorrect and using the parent strand as a template, add in the correct one. This
repairs about 99% of errors.
2) Mismatch Repair: A mechanism for repairing errors made during DNA replication,
whereby a group of proteins (enzymes) recognize a mispaired nucleotide on the newly
synthesized DNA daughter strand and replace it with a correctly paired nucleotide.

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Similar in both prokaryotes and eukaryotes.

Errors that still exist are considered to be mutations in the genome.

DNA Replication Eukaryotes and Prokaryotes:

Similarities:
- Requires Origins of replication
- Have elongation occur in the 5’ to 3’ direction
- Continuous synthesis of leading strand and discontinuous synthesis of lagging strand
- Require use of a primer for synthesis of the Okazaki fragments of the lagging strand
- Use DNA Polymerase enzymes

Differences:
- Faster replication in prokaryotes. Due to eukaryotes having more elaborate enzyme
complexes and more stringent proofreading mechanism
- Different DNA polymerase enzymes and different number (5 in proks/13 euks)
- Single origin of replication in the smaller circular chromosome of a prokaryote versus
eukaryotes may have thousands of origins of replication in the large linear
chromosome.

Telomeres: a repetitive section of DNA near each end of the chromosome which helps to
protect from loss of genetic information during replication of the linear DNA in eukaryotic
cells.
Telomerase enzyme synthesizes these telomeric regions and can replace a sequence that has
been lost. Telomerase activity decreases with age as tissue growth slows.
(Final RNA primer is removed from the 5’ end of the lagging strand, there is no adjacent
fragment to add nucleotides and fill in the gap due to the linear nature of eukaryotic
chromosomes and a shorter strand and shorter chromosome will be produced. Telomeres
helps to ensure genetic information is not loss).

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Chapter 6: Gene Expression

Section 6.1: The Transform of Information from DNA

Link between Genes and Protein:

English Chemist and Physician, Archibald Garrod (1857-1936) published his studies in 1902 of
patients with a disease (alkaptonuria) which causes urine to turn black when it is exposed to
air due to increased levels of homogentisic acid in the urine.
Correctly proposed that the build up of acid was a result of a defective enzyme in the
metabolic pathway that breaks down the amino acid phenylalanine. Hypothesized it was
inherited disease when he found that patients were blood relatives. Suggested the black urine
phenotype was due to a recessive inheritance factor and resulted in a defective enzyme.

1941- George Beadle and Edward Tatum from Stanford University looked for evidence of a
relationship between genes and enzymes.
Not known whether the production of all enzymes in a biochemical pathway where controlled
by one gene or one gene controlled the production of one enzyme.
Used a bread mold- Neurospora crassa.

- Normal (wild-type) N. crassa grow on minimal medium with sugar and inorganic salts
which is the only nutritional substances needed to synthesize the biochemical
compounds that it needs to grow.
- Created mutant strains by exposing them to x-rays (known to cause changes to genes).
- Some strains could no longer grow unless they were given additional nutrients.
- Isolated mutant strains that only grew when growth medium was given arginine
(amino acid)
- Hypothesized: If there is a one-to-one relationship between a gene and an enzyme
then, a defective gene will produce a defective enzyme. A defective gene in one of the
steps would stop the production of its intermediate compound and there would be no
growth unless it was added to the medium.
- Isolated mutant strains defective at one step in the arginine synthesis pathway and
could determine the step that was affected by each mutation.
- Found 4 genes that produced a certain enzyme that catalyzed the production of an
intermediate.
- Concluded that one gene specifies one enzyme called the one-gene/one-enzyme
hypothesis
- Now called one-gene/one-polypeptide hypothesis: one gene codes for one
polypeptide (or protein).

Messenger between DNA and proteins:

1953 Frederick Sanger (English Biochemist) showed that proteins consist of amino acids
covalently linked together. Using insulin, he established that each molecule was made up of
the same sequence of amino acids.
Notes

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• Eukaryote genes were known to be located on chromosomes in the nucleus.

• Protein synthesis occurred in the cytoplasm only.
• Some evidence to support an intermediary nucleic acid, RNA between DNA and
protein as RNA could be found in both cytoplasm and nucleus. RNA concentration also
correlated with protein level production. Also, knew that RNA was synthesized in the
nucleus and transported to the cytoplasm

1961- Francois Jacob and Jacques Monod hypothesized that there was a special type of RNA
that acts as a genetic messenger.
Proposed that this was synthesized from the DNA of genes and its base sequence would be
complementary to the gene DNA sequence and would provide the amino acid sequence info.
needed for protein synthesis.

Messenger RNA (mRNA): RNA that contains the genetic information of a gene and carries it
to the protein synthesis machinery; it provides the information that determines the amino
acid sequence of a protein.
1964- Sydney Brenner, Matthew Meselson and Francois Jacob confirmed its existence.
Messenger RNA Hypothesis:
- Bacteria infected by a virus, specific virus RNA molecule was synthesized and became
associated with bacterial ribosomes.
- New RNA molecule had a base sequence complementary to the DNA (carried genetic
info to produce the viral protein)
- Viral RNA molecule was newly synthesized and not part of the ribosomes.

Genetic Code: a set of rules for determining how genetic information in the form of a
nucleotide sequence is converted to an amino acid sequence of a protein; a code specifying
the relationship between a nucleotide codon and an amino acid.

Triplet Hypothesis: a proposal that the genetic code is read three nucleotide bases at a time.
(there were not enough possible combinations with less than 3)

1961- Francis Crick and Sydney Brenner

used T4 Bacteriophages (viruses that infect
E.coli) and made a series of viral strains
where the DNA sequence for the specific
viral protein was altered.
Found when they added or deleted one
nucleotide (or a pair), the protein was not
produced. Could be reversed if more
mutations could introduce the triplet codon
needed.
Showed that the code is read in triplets and
continuously.

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1961-1965- Various researchers worked on determining which codons specific amino acids by
comparing artificially synthesized RNA molecules of known nucleotide sequences with the
amino acid sequences of polypeptides.

Genetic code is always read from the mRNA codon rather than the nucleotide sequence of the
DNA
Characteristics of the Genetic Code:
1. Genetic code is redundant: More than one codon can code the same amino acid.
2. Continuous- knowing where to stop and start is essential as there are no pauses. A
shift will result in incorrect amino acid sequences.
3. Nearly Universal- Almost all organisms build proteins with the same genetic code ( a
few species of protists are the exception to this).

Gene Expression: the transfer of genetic information from

DNA to RNA to protein.

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Central Dogma: the theory that genetic information flows

from DNA to RNA to protein.

Transcription: the synthesis of RNA from a DNA template.

(mRNA based on the DNA template)

Translation: the synthesis of protein from a mRNA

template. (based on the mRNA sequence).
Translation uses the genetic code.

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Section 6.2: Transcription: Synthesizing RNA from DNA

RNA- polymer of nucleotides

- Four nucleotides (AUCG)
- Single stranded (can fold
back on itself)
- Complementary base pairing
(stabilizes looped structure)
- Sugar is a ribose
- Synthesized in the nucleus
- Different types of RNA
molecules

Transcription:
Objective: accurately produce a copy of a small section of genomic DNA.
Three defined stages. Very similar in prokaryotes and eukaryotes. Main difference is the
proteins involved (more in eukaryotes) and more complex associations.

Initiation:
For each gene, only one strand of the double stranded DNA
molecule is transcribed called the antisense strand or
template strand
Sense strand/coding strand: the strand that is not
transcribed. It will have the same sequence as the product
mRNA with T instead of Us.
In a single DNA, either strand can be the sense strand.
RNA Polymerase: the main enzyme that catalyzes the
formation of RNA from a DNA template.
RNA polymerase complex bind to the DNA molecule, it
unwinds and opens a section of the double helix

RNA polymerase bind to the Promoter region on DNA:

sequence of nucleotides in DNA that indicates where the RNA
polymerase complex should bind to initiate transcription.
-two sets of sequences to ensure that RNA polymerase binds
correctly and in the correct orientation to ensure that it is
copied correctly.

Elongation:
RNA polymerase complex moves along the DNA molecule, synthesizing a mRNA that is
complementary to the template strand of DNA.
Work in the 5’ to 3’ direction, adding each nucleotide to the 3’ OH group of the previous
nucleotide.
Only transcribe one strand of the template DNA (no need for Okazaki fragments).

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As soon as one binds and moves along the DNA, a second can bind and synthesize another
mRNA. (do this faster than DNA polymerase does so can synthesize hundreds of copies from
one gene at one time).

RNA polymerase does not have a proofreading function. Errors only occur in one protein and
not the genetic makeup, therefore it is better to be able to make more.

Termination: specific nucleotide sequence signals transcription to stop.

RNA polymerase stops when it reaches this and detaches from the DNA.
New mRNA releases and DNA double helix reforms

mRNA Modification in Eukaryotes:

Prokaryotes, mRNA can go onto translation as soon as transcription is done (they occur at the
same time)
Eukaryotes, modifications must occur which convert precursor mRNA (pre-mRNA) mRNA that
has not undergone processing to mature mRNA mRNA that has undergone processing.

1. Addition of a 5’ cap-
covalent linkage of a
modified G nucleotide
to the 5’ end of the pre-
mRNA. (which can be
recognized by the
protein synthesis)
2. Addition of a 3’ poly-A
tail- covalent linkage of
a series of A
nucleotides added to
the 3’ end of the pre-
mRNA which makes it
more stable and it can exist longer in the cytoplasm
3. Removal of introns: introns- non-coding regions and exons- coding regions.
Splicing- a process of excising out the introns and combing with the exons help form
the mature RNA.
snRNPs (composed of a snRNA and proteins)
recognize regions where exons and introns
meet and bind there. From interactions with
other proteins they form a spliceosome
complex that removes the introns. Alternative
splicing is when only certain exons are used
to form the mature mRNA and allows for one
gene to code for more than one protein (cell
can produce a form of protein specific to that
cell)

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Section 6.3: Translation: Synthesizing Proteins from mRNA

Second stage: translate nucleic acid code of mRNA into the amino acid code of a protein.

See Table 6.4 for overview of main components needed.

Transfer RNA (tRNA): an RNA molecule that links the codons on mRNA to the corresponding
amino acid for protein synthesis.
-single stranded RNA
-folds into 2D cloverleaf shape. Stem loops are double-stranded RNA through intramolecular
base pairing.
-Two functional regions:
Anticodon loop: a triplet of bases positioned at one end of a tRNA that recognizes and base-
pairs with a codon on mRNA during protein synthesis. It is complementary to a specific mRNA
codon.
Acceptor stem: the 3’ end of a tRNA molecule that is the site of attachment for a particular
amino acid based on the anticodon.
Aminoacyl-tRNA synthetase: enzyme responsible for attaching an amino acid to a tRNA
according to its anticodon.
Example: mRNA codon is CGG, the tRNA has an anticodon that is 3’-GCC-5’ which carries the
amino acid arg.

Ribosomes: cell structure composed of proteins and rRNA that provides the site where
protein synthesis occurs. Provide the place where mRNA and tRNAs with amino acids, and
enzymes interact and assemble.
rRNA: the RNA that is associated with proteins in the ribosome.

Each ribosome has two subunits. Small and large composed of different proteins and rRNA
molecules.
Ribosome has binding site for mRNA and 3 for tRNA. This allows for complementary base-
pairing between tRNA anticodons and mRNA codons.
Several ribosomes are often attached and translating a single mRNA (as soon as one has
moved down the mRNA another one will attach).

Polyribosome: a structure composed of multiple ribosomes along a strand of mRNA. Several

copies of polypeptide at the same time.

-One of the most energy consuming processes of the cell due to the complex nature of the
steps, components and the energy needed to bind 2 amino acids together.

Step One: Initiation

-Initiation factors (proteins) assemble the small submit unit (ribosome), mRNA, initiator tRNA,
and the large subunit.

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-small subunit attaches to the mRNA near the Start codon: a triple of three bases that
specifies the first amino acid of a protein. (AUG).
-First tRNA that binds to the codon is the initiator tRNA with a UAC anticodon. This is met or
methionine
-large subunit joins to form active ribosome.
-start codon sets the reading frame for the
gene.
Reading Frame: the codons of mRNA that are
read to produce an amino acid sequence and
is set by the start codon.

-3 binding sites for tRNA = P (peptide) site

with the tRNA and the growing polypeptide
attached; A (amino acid) site the tRNA with
the next amino acid to be added, E (exit) site
where the uncharged tRNA which no long has
the amino acid is ejected.

At initiation, the initiator tRNA binds to the P site.

Elongation: PROTEIN SYNTHESIS

-polypeptide becomes longer, one amino acid at a time
Elongation factors enable the tRNA anticodons to bind to the mRNA codons.
(Once the initiator is bound to the ribosome), A site is occupied by the tRNA with an
anticodon that base-pairs with the second mRNA codon and the second amino acid.
Peptide bond: a covalent bond formed between two amino acids during protein synthesis.
Forms between the two amino acids
Dipeptide is attached to the tRNA at the A site.
mRNA moves along by one codon and this
becomes associated with the P site of the
ribosome.
These four steps are repeated rapidly.
1. tRNA with attached polypeptide is in the P
site. tRNA with the next amino acid enters
the A site.
2. Polypeptide is transferred to the amino
acid on the A site.
3. mRNA moves forward by one codon.
Polypeptide tRNA is now at the P site.
4. Uncharged tRNA exits. New codon is at the A site and can receive the next tRNA wit
the amino acid.

Termination:
Stop codon is reached on the mRNA. Polypeptide and the translation machinery are
separated.

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Release factor (protein) cleaves (cuts) the

polypeptide from the last tRNA
Polypeptide released and will fold into a 3D shape.

Review:
Expression of a gene: refers to synthesis of a protein
that is encoded by that gene.

Transcription: DNA sequence of a gene serves as a

template for the synthesis of mRNA. In eukaryotes
processing of the mRNA occurs (remove of introns,
adding 5’cap and 3’ poly-A tail).
Translation: mRNA carries genetic information to
the ribosome where protein synthesis occurs. tRNA molecules bring amino acids to the
ribosomes. Order of the
codons on the mRNA
determines the order that
the tRNA amino acids bind
and then determines what
the polypeptide will form.

Mutations and Effects of

Mutagens:
Mutation: a permanent change in the nucleotide of a cell’s DNA. All mutations are copied
during DNA replication and passed to daughter cells.
Mutations in daughter cells can be passed generations. Those in somatic cells do not.
Most are harmless, rarely are they beneficial (lead to species change and adaptation).

Two types:
Single-gene mutation: mutation that involves changes in the nucleotide sequence of one
gene
Chromosome mutation: mutation that involves
changes in chromosomes and may involve many genes.

Single-Gene Mutations:
Point Mutation: mutation involving a single base pair
substitution, insertion or deletion.
Substitution may be fairly minor effect due to the
redundancy of the genetic code.
Change in the coding sequence of a gene does not
necessarily result in a change in the polypeptide. Ie. CCT to CCC has mRNA codons GGA and
GGG both which code for glycine.

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Frameshift Mutation: mutation caused by addition or deletion of a number of nucleotides

not divisible by 3, resulting in a change in the reading frame.

Affecting the amino acid sequence:

Silent Mutation: a mutation that does not
change the amino acid sequence of a
protein.
Missense Mutation: a mutation that
changes the amino acid sequence of a
protein.
Can be harmful.
Nonsense Mutation: mutation that
shortens a protein by introducing a stop
codon. Usually harmful.

Chromosome Mutations:
Changes to chromosome number of
the genome are always detrimental
and lethal.
Also involve rearrangement of
genetic material and can affect
several genes even those on
different chromosomes.
Changes include deletion or
duplication of portions of
chromosomes.
Inversions: chromosome segment is
broken and re-inserted in the
opposite direction.
Translocation: one chromosome section is broken and used to another chromosome.

Many spontaneous mutations occur naturally in the cell. Rate varies among organisms and
even among different genes in one cell.
Example: Incorrect base pairing by DNA polymerase during replication
DNA Transposition can disrupt extensive regions of genetic information and involves the
movement of specific DNA sequences within and between chromosomes.
Transposons: a short segment of DNA capable of moving within the genome of an organism,
also called a jumping gene.

Exposure to certain factors in the environment can increase mutation rate.

Induced: mutations caused by agents outside the cell.
Mutagen: substance or even that increases the rate of mutation in an organism.
Two types:

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Physical Mutagen: Physically change he structure of DNA. Examples:

-X-rays (high energy radiation)
-UV radiation (reaction between Adjacent pyrimidine bases (C and T) and distorts DNA
affecting replication

Chemical Mutagens: molecule that can enter the nucleus

of a cell and induce mutations by reacting chemically
with DNA. May cause a frameshift mutation or a
nucleotide substitution.
Can cause incorrect nucleotides to be inserted.
Examples: Nitrites (cured meats), gasoline fumes,
cigarettes.
Most are carcinogenic.

DNA Repair:
-DNA polymerase can repair incorrect nucleotides during
replication which helps to reduce replication errors.
Specific proteins that act to recognize errors and repair the damage.
Photorepair (specific repair): mechanism for repairing thymine dimer structures from UV
radiation which causes 2 adjacent Ts to be covalently linked together forming a dimer.
Excision repair (non-specific repair): removing damaged region of DNA and replacing it with a
correct sequence.
Section 6.4: Regulation of Gene Expression

Gene Regulation: refers to the control and change to gene expression in response to different
conditions in the cell or environment.
Includes whether gene is active or inactive but also determines level of activity and the
amount of protein that is available in the cell.

Constitutive gene: a gene that is constantly being expressed; it does not undergo regulation
of expression (essential for cell survival) a.k.a housekeeping genes

Most genes are regulated so that the protein is only expressed at certain times and amounts

Prokaryotes Gene Expression:

Three different levels: during transcription, translation, and after protein is synthesized.
Most common: level of transcription, during initiation
Most studied system: E. coli

Operon: a cluster of genes grouped together under the control of one promoter; occurs in
prokaryotic genomes
Genes in the same metabolic pathway are often found in the same operon. Genes are
transcribed together into one continuous mRNA strand called a polycistronic mRNA.
Individual proteins are made from the one strand.

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The lac Operon:

E. coli uses different sugars as a source of energy and carbon and can adjust its gene
expression based on what sugar is available. Ex. Lactose present = increase in enzymes that
metabolize lactose; lactose absent = decrease in enzyme levels

Genes that encode enzymes to break down lactose are found in the lac operon on the
chromosome.

-consists of a coding region and a

regulatory region
-Coding region has genes for 3 enzymes
that are needed for the breakdown of
lactose
-Regulatory region has the promoter for
transcription of lactose enzymes. Also
contains sites to regulate transcription:
operator and a Catabolite activator
protein (CAP) binding site

Operator: a DNA sequence element to which a (repressor) protein binds to regulates

transcription
Repressor: the protein that binds to a particular DNA sequence (operator) to regulate
transcription; inhibit the transcription of a gene(s)
CAP binding site: DNA sequence where a specific protein binds. Binding to CAP increases the
rate of transcription of a gene(s)
Activator: a protein that binds to a particular DNA sequence to regulate transcription;
increases transcription rate of a gene(s).

a) Lactose absent: repressor protein

binds to the operator which stops RNA Polymerase from binding to the promoter.
TRANSCRIPTION CANNOT OCCUR.
b) Lactose present: Allolactose (derivative) is produced and binds to the repressor which
now cannot bind to the operator. TRANSCRIPTION OCCURS.
Binding of CAP activator protein will increase transcription levels.

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-called an inducible operon; transcription is induced when lactose is present.

Trp Operon: Similar structure to the lac

operon. Coding region has 5 genes for
enzymes needed for the synthesis of the
amino acid tryptophan.
Regulatory region has a promoter and
operator region.
Normal conditions; Trp must be made so
the repressor does not bind to the operator
and transcription occurs.
When Trp reaches a certain level, some of it
will bind to a repressor protein which
increases its ability to bind to the operator
and transcription decreases.

Gene Expression Regulation in Eukaryotes:

-more complex, more levels
-5 levels: pre-transcriptional, transcriptional, post-transcriptional, translational, post-
translational

Pre-Transcriptional and Transcriptional Control:

-DNA is associated with proteins such as histones to form nucleosomes which assemble into
more condensed structures to form chromatin.
-DNA in highly condensed areas of chromatin is NOT transcribed because chromatin is a
physical barrier to proteins needed to synthesize pre-mRNA.
-chemical modifications of the histone proteins and the use of multi-enzyme structures called
chromatin-remodeling complexes will alter chromatin structure and loosen nucleosome
structures so that proteins for initiation of transcription can gain access.

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-Each gene has its own promoter and transcription

control is distinct for that gene.
-Transcription Factors: one of a set of proteins required
for initiation of transcription; required for the RNA
polymerase complex to bind to the promoter.

-transcription factors must interact with the promoter

for RNA polymerase to initiate.
-activator proteins increase transcription initiation by
binding to transcription factors and RNA polymerase as
well as enhancers: specific DNA sequences.
-many activators (type and number) and gene
regulation usually needs more than one type.

Post-transcriptional and Translational Control:

-alternative splicing of the mRNA produces different mRNA molecules and different protein
products
-Modifications can be altered so that the cap and/or tail are NOT added.
-mRNAs will either not be transported from the nucleus or undergo rapid degradation in the
cell. Therefore, NO mRNA is available to make a protein

RNA Interference: the regulation of gene expression by small RNAs; inhibits gene expression
by degrading mRNA or inhibiting translation. Two are: micro RNA (miRNA) and small
interfering RNA (siRNA). Associate with protein complexes and turn off gene expression by:
promoting mRNA cleavage or inhibiting translation. They do this by forming complementary
base pairs with the mRNA.

Post-Translational Control:
-polypeptides synthesized are not necessarily active and must undergo modifications. Ex.
Insulin is initially folded into a 3D structure but to be active, amino acids are removed leaving
2 polypeptide chains. Covalent bond forming between 2 sulfurs on each chain will join them
and activate the protein. Also, covalent linkage of a phosphate group to one or more amino
acids can also make the protein functional.
-regulation can also include how long protein is available for. Ex. Attaching a chain of
ubiquitin molecules to a protein which is a signal for protein degradation.

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Chapter 7: Genetic Research and

Biotechnology
Section 7.1: Techniques for Producing and Analyzing DNA

Molecular Biology: the study of the structure and functions of nucleic acids and proteins.

Recombinant DNA: a molecule of DNA composed of genetic material from different sources.
It has been prepared in the laboratory by adding fragments together usually from genomes of
different species.
How did this become possible?
Due to isolation of a certain type of enzyme from bacteria called restriction enzymes which
help prokaryotes defend themselves against infection from foreign viruses. The enzymes stop
the replication of infecting viruses by cutting (cleaving) viral DNA.

Restriction Endonuclease: an enzyme that cleaves the interior (rather than the end) of
double-stranded DNA in a sequence-specific manner.
How?
They recognize a short nucleotide sequence called a target sequence in the DNA. The enzyme
will cut the strand at a certain point in the sequence known as the restriction site.
Different endonucleases recognize different target sequences.
Restriction fragment: a small segment of DNA generated by cutting a larger piece of DNA
with a restriction enzyme.

Two characteristics that make them useful:

1. Sequence Specificity: Cuts are specific and
predictable.
2. Staggered cuts: most produce a staggered cut that
leaves a few unpaired nucleotides on a single
strand at each end of the restriction fragment
called sticky ends or overhangs. They can form
base pairs with other single stranded regions with
a complementary sequence.
Also produce blunt ends that allow any two fragments of DNA with blunt ends to be
combined. Loss of specificity also means that many by-products can form, which makes the
process less efficient.

This process is done in the laboratory in reaction tubes with volumes in microlitre amounts.
Often to make many copies of a specific DNA segment such as a gene.

Steps to make a recombinant DNA molecule.

1. Select restriction endonuclease to cut both DNA fragments to be combined.

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2. React each DNA piece with the enzyme to make

DNA fragments.
3. Incubate the fragments with DNA Ligase to seal
the fragments, forming covalent bonds.
Gene Cloning (in Bacteria):
Gene Cloning: the process of manipulating DNA to
produce many identical copies of a gene or another
segment of DNA in foreign cells.

Why? To study the gene itself or to use the gene to

produce RNA and/or protein in enough amounts to study these molecules.
Bacteria are used as host systems because they are 1) straightforward 2) inexpensive 3)
grown easily 4) large amounts 5) reactants have been optimized for use in E. coli.

Steps:
1. Produce a recombinant DNA molecule with the
gene to be cloned and a vector (carrier for the
gene) for the host system. Plasmid acts as a vector
for gene to be cloned.
Plasmid: self replicating, closed circular piece of
DNA that can act as a carrier of a gene to be
cloned in bacteria.
(small circular double-stranded DNA not part of
the nucleoid region, often have non-essential
genes (may carry genes for antibiotic resistance)).

Plasmid has an origin of replication so it can be

copied separate to the bacteria’s chromosomal
DNA.

Notes
• It must carry a gene to make the bacteria
resistance to a certain drug (antibiotic
ampicillin).
• It must have restriction endonuclease
site(s) to insert the gene to be cloned.
• Often DNA to be cloned interrupts the lacZ gene that codes for an enzyme that breaks
down galactose.
• Ampicillin and lacZ are called selectable markers as they can be used to select for the
bacterial colonies that contain the recombinant DNA of interest (from those that have
plasmids but not the DNA inserted).
2. Introduce the reaction mixture into the bacteria.
Transformation: process in which a bacterial host takes up a segment of DNA from the
environment under particular experimental conditions.

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Treat bacterial cells with chemicals to make the cell membrane permeable to the DNA
Transformed bacterial cells are those that take up the DNA.
3. Place the bacterial cells in a petri dish with the growth media and 1) ampicillin and 2) derivative of
galactose, X-gal. Cause bacterial colonies to turn blue (when it has been broken down by the
enzyme coded for the lacZ gene).
4. Identify bacterial colonies with recombinant DNA cells. All colonies that grow on the dishes with
ampicillin must have either the recombinant DNA or the plasmid. Also, all the blue colonies
contain an active lacZ gene and do not have the gene in them. The white colonies have the
recombinant DNA.
5. Select the cells from the colonies with the recombinant DNA. Grow them in liquid culture to make
more.
6. Isolate the recombinant DNA molecules and purify them from the bacterial cells.
7. Use analysis techniques to ensure that the correct recombinant DNA has been made.

DNA Amplification: the process of producing large quantities of DNA from a sample.
For gene cloning, the recombinant DNA molecule is transformed into E. coli cells and grown in
liquid media to produce large cultures in which the molecule can be isolated and purified.

Alternative method to amplify only a small section of DNA that does not rely on recombinant
DNA molecules or host systems.

Polymerase Chain Reaction

(PCR): an automated method
for amplifying specific regions of
DNA from extremely small
quantities.
Highly specific and rapid
involving repeated cycles of the
same basic steps to produce
copies of the DNA.
Steps:
1.DNA sample is heated to 95⁰C
(high temp) to denature the
DNA into single strands.

2.DNA sample is cooled with 2 nucleotide primers that are complementary to each 3’ end of
the DNA fragment (to be amplified). Low temp. allows the primers to anneal or base pair with
the 3’ ends of the single stranded DNA. Temp. is about 55⁰C.

3.DNA sample is heated to 72⁰C (optimal temp.) so Taq polymerase can synthesize DNA by
adding free nucleotides to the ends of the primers by complementary base pairing.
Taq polymerase is a type of DNA polymerase that can withstand high temps. and was isolated
from heat-loving bacteria (Thermus aquaticus).

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4.Steps 1-3 are repeated several times- denaturation, primer annealing, DNA synthesis from
primers.
DNA amount doubles with each replication cycle. (1฀2฀4฀8)

What is it used for? Gene of interest in gene cloning, compare DNA extracted from
mummies/fossils, history of human populations, screen for genetic defects in early embryos,
small sample found at crime scenes.

Gel Electrophoresis: tool used to separate molecules according to their mass and charge; can
be used to separate fragments of DNA.
Uses an electric field to separate negatively charged DNA fragments based on size as they
move through the gel.
Gel is submerged in buffer- aqueous solution with various salts that keep the pH of the
solution.
One end is a positively charged anode, other is a negatively charged cathode.
Gel is made of either polyacrylamide or agarose.
Provides a porous matrix-like support for the DNA
molecules to move- repelled by the negative
cathode, attracted to the positive anode.

Step 1: Negatively charged dye turns DNA sample

blue. Chemical ethidium bromide added to the gel
which associates with the DNA and fluoresces under
UV light.
Samples are added to wells at one end of the gel.
Step 2: Gel is put in a buffer. Power is turned on.
Electric current runs between the cathode to the
anode, through the buffer and gel.
DNA fragments move toward the +ve anode (because DNA is -ve due to phosphate groups).
Step 3: Smaller DNA fragments move quicker through the gel than larger fragments. DNA
fragments separate according to size.
Step 4: Gel removed from buffer, exposed to UV light. DNA bands can be observed.

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DNA fingerprinting: used to identify individuals by

analyzing the DNA sequence of certain regions of
their genome. A.k.a DNA profiling
DNA of an individual is unique (only exception is
identical twins)
Restriction fragment length polymorphism (RFLP):
Treat chromosomal DNA with restriction
endonucleases and separate them using gel
electrophoresis.
Recent method: Short tandem repeat (STR)
profiling: used to identify individuals based on
repeating short sequences of DNA in the genome
that vary in length between individuals.
STRs= repeating short sequences of DNA in the
genome and vary in length between individuals depending on how many copies of a certain
STR are present.

Notes
Many different loci (locations of DNA sequences/genes) of STRs can be analyzed. STRs are
amplified using primers and PCR and separated by gel electrophoresis. More repeats at an
STR locus, the longer the DNA fragment and the shorter it will travel. The fragments are
fluorescently labelled and a detector measures the emittance of each STR.
DNA fingerprint is a pattern of peaks with certain molecular masses.

Uses? Crime scene, database of human STR profiles, identify victims, parentage

DNA Sequencing: method for determining the nucleotide sequence base by base of a
fragment of DNA.
Manual DNA Sequencing:
Maxam-Gilbert Sequencing (1976): uses radioactive labelling of a single-stranded DNA to be
sequenced. DNA is cleaved at specific bases producing radioactively labelled fragments that
are separated by gel electrophoresis.
Dideoxy sequencing (1977): method for determining a sequence
of DNA fragment using dideoxynucleotides which cause
termination of DNA synthesis. A.k.a dideoxy chain termination
method.
DNA polymerase makes a series of DNA fragments of different
lengths, using the DNA to be sequenced as a template.
Fragments all start at the same position but end at different
specific bases because replication terminates/stops when 1 of 4
dideoxynucleotides (ddA, ddG, ddC, ddT). They lack a hydroxyl
group at the 3’ and 2’ carbons of the ribose sugar and cannot
react with a new nucleotide (missing at 3’ end).

Steps:

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1.Denature the DNA to single stranded. Primer anneals to the 3’ end of the region.

2.Four separate reactions prepared.

Each has single stranded DNA, primer,
deoxynucleotides, DNA polymerase, 1
of 4 dideoxy nucleotides.

3.NA synthesis proceeds. Series of

fragments of different lengths will be
made in each reaction. Fragments are
complementary to the template.

4.Each of the 4 reactions is separated

using gel electrophoresis with
polyacrylamide gel allows for high
resolution of fragments that differ by
only one base. Radioactive tag attached to the dideoxy nucleotides to visualize the DNA and
expose the gel to x-ray film called autoradiography.

4. Gel read from top to bottom (longest-shortest fragment). By reading which base is at
the end of each strand and comparing the lengths, the nucleotide sequence of the
original DNA is formed.

6. Sequence of the original template is the complement to the synthesized strand.

Human Genome Project: project that sequenced the human genome and identified all the
genes within it.

DNA sequencing allowed scientists to determine the nucleotide sequence of genes and thus
amino acid sequences of the proteins.

Manual sequencing was time consuming (only about 300 base pairs can be read at any one
time).
Automated sequencing was developed to run in less time and at once. Early methods used
dideoxynucleotides with dye tags that can be added to one tube can run out on a single lane
in a gel which reduce the time to separate fragments. Laser scans cause the dyes to fluoresce,
each one a different colour. Computer printout of coloured peaks is made.

Since 2005, new techniques “next-generation sequencing” improving data output 10 fold.
Why does it improve?
• medicine- individualized diagnoses and treatments to determine risk of disease or
treatment option
• cancer diagnosis and treatment- Tumour profiling determining DNA sequence of a
tumour.
Must be affordable and take less time.

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Site-directed mutagenesis: a method of specifically altering the nucleotide sequence of a

region of DNA.
Showed that specific mutations could be made in the DNA sequence of a gene and could
target specific sequences in genes.
Now can alter codons in a corresponding gene to create a form of the enzyme with an altered
amino acid. One can study the activity of the mutant enzyme.

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Section 7.2: Production and Regulation of Genetically Engineered Organisms

Gene Therapy: method for treating genetic disorders by introducing a correct form of the
disease-related gene into an individual’s genome.
Ability to alter human genome has given rise to serious concerns, i.e. creating “designer
babies” and the genetic manipulation of animals for sale as pets i.e. Glofish.

Genetic Engineering: alteration of genetic material of an organism in a specific manner.

Involves precise changes to DNA sequences such as introducing a mutation into a gene,
introducing foreign DNA (i.e. gene from another species).

Transgenic: an organism that is produced from the introduction of foreign DNA into its
genome providing it with a new phenotype.

Genetically modified organisms (GMOs): organism whose genetic material has been
modified often through the insertion of a foreign gene into its genome.

Biotechnology: use of an organism or a product from an organism for the benefit of humans.
Using tools of molecular genetics to produce organisms and their products.

Patents: a government ruling giving an individual or organization the sole title or right to
make, use or sell a particular invention.

Private companies that use recombinant techniques to produce GMOs want to claim
ownership of the organism and its genome.

Applications:
Transgenic Bacteria in Pharmaceuticals:
Recombinant DNA technology makes transgenic bacteria using a specialized plasmid vector
called an expression vector: plasmid vector that is transformed into a
host cell for the purpose of producing foreign protein.

Why is it needed?
vector must have specific sequences to support both transcription and
translation of the introduced gene for which the recombinant molecule
codes.

First successful example was producing insulin.

Human insulin has 2 polypeptide chains (A and B) which are made in
separate bacteria batches. Expression vectors have the DNA sequence for
the gene for the A or B chain as well as the gene that codes for a bacterial
beta-galactosidase enzyme.

Results in large amounts of fusion proteins made of the enzyme fused to

either chain. This protects the chain from being degraded by the bacteria.

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The protein is purified and the chain is removed. The two chains are then mixed together and
allowed to properly fold and make insulin.

Health Canada approved the sale of transgenic insulin in 1983. Prior to this date, those with a
defect in the beta cells of the pancreas had to use insulin from animal pancreas sources
(similar to human form) which is expensive and labour-intensive. Many people had allergic
reactions and opposed this due to religious reasons.
Other examples: human growth hormone, hepatitis B vaccine, erythropoietin.

Bioremediation: use of micro-organisms or other living cells for environmental clean-up

Micro-organisms convert environmental toxins into non-toxic pollutants. Ex. Bacteria strain
that can naturally break down petroleum (crude oil).
Others break down pesticides and herbicides that have been released into the water or
remove sulfur from coal to produce cleaner emissions.

Transgenic Plants:
Manipulation of plants has occurred for long time from the slow process of crossbreeding
different varieties to produce plants with desired traits.
Most prominent examples of GMOs are agricultural plants and those made for human
consumption. Ex. Soybeans, tomatoes, corn, canola (most common in Canada), potatoes.
Genetic modifications include increased tolerance to herbicides, greater resistance to disease
and pest infestations.
Benefits: increased crop yield, reducing harvest cost, reducing pesticide use, increased
nutritional value, increased food quality, slower spoilage

Totipotent: one cell can grow and divide to produce all of the different types of cells in a
plant. Therefore, a gene can be introduced into somatic tissue (i.e plant leaf) and produce a
transgenic plant as the leaf is treated with plant growth hormones to form roots and shoots
and then whole plant.

Two methods to introduce foreign DNA into plant cells:

Biolistic Method: involves bombarding plant cells with particles coated in DNA that become
integrated into the plant genome. Aka gene-gun method involves striking plant cells with tiny
particles of gold or platinum coated in DNA at a high speed so the DNA can penetrate the cell
wall. However, lacks control with insertion site and how many copies are introduced. As well,
the introduced gene could insert into a functional gene and alter the gene’s function.
Ti Plasmid Method: uses the tumour-inducing plasmid from Agrobacterium tumefaciens as
the vector for the insertion of a foreign gene into a plant genome.
Bacteria naturally infect plant cells causing bulbous growth on the plant. Part of the Ti plasmid
called the T-DNA integrates into the plant genome and causes uncontrolled cell growth

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resulting in a tumour. Alteration of

the T-DNA no longer causes tumour
formation but allows for the DNA to
be integrated.
Steps:
1.Recombinant DNA molecule is
produced (gene of interest is inserted
into the T-DNA region of the Ti
plasmid) with a selectable marker resistance to antibiotic kanamycin.
2.Recombinant Ti plasmid is taken up by the bacterium
3.Plant cells are infected with the bacterium and the recombinant DNA with the gene of
interest integrates into plant cells.
4.Selectable marker used to determine which cells have the recombinant DNA. Ones that
survive when exposed to the antibiotic kanamycin have it. Antibiotic will kill cells of the
bacterium so only plant cells remain. Transgenic plant can be grown from these cells.

Controversy:
1. Are they safe for human consumption?
- Each crop requires approval, 7-10 years of health and safety research before they can be
consumed. Some reports of allergic reactions reported. Some argue not enough time to see
long-term effects.
2. Will GM Crops have negative impact on the environment?
- Risk of gene transfer after being introduced into a crop.
Two types of transfer: Horizontal gene transfer: introduced trait is transferred to other
organisms such as plants, animals, bacteria, fungi. This transfer occurs at a very low rate in
nature.
Vertical gene transfer: transfer of gene or trait into genomes of natural or wild versions of
the same crop. No evidence so far but is monitored.
- Potential harm for those that express a toxin.
o Example. Bt corn contain a that is toxic to many insects. Issue of
resistance of insects to the Bt corn but regulations try to reduce this
such as planting both Bt corn and non Bt corn.

Transgenic Animals:
Foreign gene is inserted into the genome of an animal oocyte (egg) that is fertilized.
Fertilized egg is implanted into a host female and develops. The offspring is a
transgenic form. Procedure has been used to produce transgenic fish, pigs, cows,
rabbits, sheep. Can be produced with improved traits such as pigs that can break
down phosphate more efficiently and reduce excretion and runoff into water
sources.

Gene Pharming: process of using livestock to produce therapeutic proteins

(pharmaceuticals).
Involves protein production in the mammary gland of the transgenic animal. Human protein is
excreted in the milk, collected and purified. Foreign protein is targeted to the mammary cells

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through a beta-lactoglobulin promoter in the recombinant DNA containing the gene that
encodes the protein.
Example: treating hemophilia and emphysema are two successes.

More expensive and difficult than using bacteria but proteins are not always produced in
proper form in bacteria as they undergo modifications after expression in human cells which
cannot occur in bacteria. Many foreign proteins are also degraded in bacteria.

Cloning of Mammals from Somatic Cells:

Natural methods include identical twins (same genome as from
same fertilized egg). First successful in Dolly in 1996. Other species
include cows, pigs, mice, dogs, cats.
Human cloning is seriously debated from being morally wrong,
threatening family life to a viable reproductive alternative for
infertile couples.

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