Unit 3 - Molecular Genetics

DNA STRUCTURE
DNA - molecule that encodes genetic information of living things
Francis Crick and James Watson are credited with discovering structure of DNA
Largely work of Rosalind Franklin that led to this discovery
DNA structure:
- Basic units that make up DNA molecules are nucleotides
- Nucleotides are made up of 3 components:
➔ nitrogenous bases
➔ Five carbon sugar (deoxyribose)
➔ Phosphate group
- dNA contains 4 different nucleotides
- Nucleotides are different due to nitrogenous bases
➔ Adenine (A) and Guanine (G) - double ringer structures known as purines
➔ Thymine (T) and Cytosine (C) - single -ringed structure known as pyrimidines
- Nitrogenous bases of one strand paired with bases in other strand: T - A (2 hydrogen
bonds) & C - G (3 hydrogen bonds)
- Known as Chargaff’s rule - if you know nucleotide sequence of one strand, you can
deduce sequence of other strands
- Base pairs held together by hydrogen bonds
- According to model proposed by james Waston and Franics Crick, nucleotides form 2
antiparallel strands
- Strand are antiparallel because they are upside down in relation to each other
- Fifth carbon in deoxyribose points upward in one strand and downward in the other
NOTE: along each strand, sugar linked together by phosphate group
➔ complementary nitrogenous base hold strands to one another through hydrogen bonds
➔ Notice that the orientation of one strand is opposite to the other (antiparallel)
- 2 strands of nucleotides are twisted into a right handed helix that makes one complete
turn every 10 nucleotides (a distance of 3.4nm)
HOW DOES IT ALL FIT?
- If DNA in one cell were laid end to end - 3m (6 billion base pairs long)
- To fit the massive amount of information into each cell, DNA is coiled upon itself to form
chromosomes
- Histones ( + changed protein) act as “spools” on which DNA molecule coils
- Nucleosomes (coil) coiled upon themselves to form chromatin form
- Chromatin condense during mitosis -seen as chromosome

DNA REPLICATION
- Before mitosis cell doubles its genetic information so each daughter Cell has identical
genetic information to parent cell
- DNA must replicate (duplicate itself)
- Free-floating nucleotides in nucleus are assembled into a copy of parental DNA
- 2 possible models to explain how this happens :
Model #1: conservation replication Model #2 : Semi - conservation replication
Model #I : Conservative Replication
- One daughter DNA molecule is constructed entirely from free-floating nucleotides
- Parental DNA molecule remains unchanged (conserved)
Model #2: Semi - conservative Replication
- Each daughter DNA molecule has one strand of parental nucleotides & one strand made from
free - floating nucleotides
- Half the parental material is conserved in each daughter DNA molecule (semi -conserved)
DNA Replication
- Matthew Meselson and franklin stani performed an experiment
- Used cultures of Escherichia Coli (E. Coil) bacteria that were given either "heavy" nitrogen (15
N) or a more common isotope of nitrogen
** Recall that Nitrogen is an essential component of DNA.
The Meselson - stahl experiment
1. Grow E. CoIl bacteria in presence of 15N only (parental DNA)
2. Take parental bacteria and allow them to reproduce in presence of 14N only (generation
I)
3. Take generation 1 bacteria & again allow them to reproduce in presence of 14N only
(generation 2)
- Bacteria that Contain DNA made of 15N have DNA "heavier" then DNA containing 14N
- When DNA is extracted from bacteria & spun in a Centrifuge samples with 15N will sink
lower in test tube than samples with 14N

DNA REPLICATION (SPECIFIES)

- Before DNA replication can occur, highly coiled and condensed DNA molecule must be
“straightened” into linear sequence of nucleotides
- Enzymes serve this function
1. as DNA uncoils and unwinds, a class of enzymes called topoisomerases relieve tension
on molecule
2. DNA helicase breaks hydrogen bonds that hold two strands together - produces a
“replication fork”
3. single stranded binding proteins (SSBP) attach to each strand to prevent hydrogen
bonds from reforming
4. RNA primase attaches RNA nucleotides (primers) to 3’ end of each strand
5. starting at primers DNA polymerase III adds complementary DNA nucleotides to each
strands
DNA polymerase III constructs new strand in 5’ to 3’ direction only
NOTICE: one strand (leading) is built continuously towards replication fork
Lagging strand is made up of short pieces called Okazaki fragments
Other strand (lagging) is built discontinuously away from replication fork

6. DNA polymerase I replaces RNA primers with DNA ligase joins okazaki fragments
7. error correction: most occurs during step 5 when DNA polymerase III is adding
complementary DNA nucleotides.
- Occasionally, DNA polymerase III will male a mismatch error
 - When this happens, the enzyme usually backs up and corrects error, before continuing
on
- 1000 nucleotides added per second bacteria
- 50 nucleotides added per second in eukaryotes
About one error in 1 million base pierced is not corrected by DNA polymerase III
Step 7: Special DNA repair complex is made up of proteins and enzymes including the new
polymerase I & DNA polymerase II remove a small section of nucleotides around error
Missing section is replaced by DNA polymerase III. Missing phosphodiester bond of the last
nucleotides is joined by DNA ligase

PROTEIN SYNTHESIS
Central dogma of genetics - DNA stores genetic code necessary for synthesizing all the different
proteins in our body
- Each protein coded for by specific segment of DNA called genes
- DNA found in nucleus and proteins are constructed by ribosomes in cytoplasm
- How do instructions get from the nucleus to ribosomes?
- Code for DNA transcribed into molecules of mRNA (messenger RNA)
- Ribosomes can translate genetic code into a protein
- mRNA moves into cytoplasm to meet ribosomes
Protein synthesis occurs into steps: transcription and translation

TRANSCRIPTION
occurs in three steps:
1. Initiation
– transcription begins "upstream" of gene, at a promoter region on the 3’ to 5’ strand
- Promoter - specific sequence of DNA nucleotides that RNA polymerase can
recognize and bind to
2. Elongation
- DNA is unwound, exposing template strand at beginning of gene
- RNA polymerase synthesizes mRNA molecule in 5’ - 3’ direction, through
complementary base pairing with DNA template
3. Termination
- RNA polymerase reaches end of gene and encounters termination sequence
- RNA synthesis stop
- mRNA and RNA polymerase are released

Post - transcriptional Modification

mRNA is modified in 3 ways before it leaves the nucleus:
5’Cap → seven methyl guanine ribonucleotide is added to 5’ end of mRNA - helps mRNA attach
to ribosome during translation
Poly - A tail → string of about 50 - 250 adenine ribonucleotides are added to 3’ end of mRNA -
products the mRNA from RNA digesting enzymes in the cytoplasm
Introns are removed → mRNA strand made of sections that code for specific proteins (exons)
and those that doesn’t (introns)
 - Proteins called spliceosomes ‘cutout’ introns, leaving only exons

TRANSLATION
Translations involves 3 different types of RNA:
mRNA (messenger) : copy if instructions for constructing a specific proteins
tRNA (transfer) : delivers individual amino acids to ribosome for construction of protein
rRNA (ribosomal) : ribosomes consist of 2 subunits - each is a combination of rRNA and
proteins
Structure of tRNA
● Single stranded and double stranded sections
● Unique anticodon
● Anticodon - 3 nucleotides segment that corresponds to codon on mRNA
● Carries a specific amino acid on 3’end (in this case tyrosine)
- 61/64 codon specify an amino acid
- 3 are stop codons (UAG, UGA, UAA)
- Fewer than 61 tRNA’s require to deliver 20 amino acids (as little as 32)
Wobble Hypothesis
➔ pairing of the anticodon with the first two nucleotides of the codon is always precise, but
there is flexibility in pairing with the third nucleotide of the codon.
Translation occurs in 3 step:
1. Initiation
- tRNA with anitcodon that corresponds to AUG (start codon) brings MET and
forms a complex with small ribosomal subunit at P site
- Complex binds to 5’ end of mRNA and scans along until it reaches AUG start
codon
- Large ribosomal subunit binds to complex ribosome
2. Elongation
- Ribosomes moves along mRNA reading codons (3 bonds)
- Next tRNA delivers amino acid to A site
- MET cleaved from tRNA
- Peptide bond formed between MET and added on RNA in A site
- tRNA with polypeptide chain moves to Psite
- Empty tRNA moves E site and as released
3. Termination
- Occurs when A site of ribosome reaches a stop codon (UAA, UAG, UGA)
- Protein release factor binds instead of a new tRNA
- Ribosomal subunits separate from mRNA
- tRNA, release factor, and polypeptide chain released

PROTEIN SYNTHESIS: CONTROL MECHANISMS

- Functions of gene is to code for protein
- Some proteins must be produced continuously, others are only needed at certain times
- Insulin, which converts glucose to glycogen, is only made when needed at certain times
- Insulin, which converts glucose to glycogen, is only made when needed (ie. after a meal)
 - Constantly producing insulin would be a waste of energy
- Cells evolved control mechanisms which regulate gene expression, by turning them
on/off depending on situation
Prokaryotic control mechanism
- E.coli - bacteria found in large intestines of mammals
- E.coli can produce an enzyme called beta-ga;actosidase, that breaks down lactose into
simple sugars (glucose and galactose)
- Since lactose is not always present, bacteria must have a method for regulating
expression of geme that codes for beta -galactosidase
- Known as lac operon
- “Lac” stands for lactose and operon is the part of bacteria chromosome that is involved
in regulations
Lac Operon
→ 3 genes that code for enzyme beta- galactosidase
→ promoter region which RNA polymerase must bind to before transcription can occur
→ operator region which overlaps with promoter region
→ Lac 1 protein can bind with operator represses transcription by preventing RNA polymerase
from binding with promoters
NOTE: when Lac1 protein is attached to operator operon is “off”, when it separates from
poetater, operon is ‘on’
Lactose
● Lactose from milk binds with the Lac1 protein causing it to change shape. With the lac 1
protein removed, RNA polymerase can bind to the promoter and transcription can occur
● While transcription occurs beta galactosidase can be produced causing the breakdown
of lactose
● When all the lactose has been broken down, the lac1 protein returns to its original
shape. Once again, RNA polymerase is prevented from binding to the promoter and
transcription stops
Eukaryotic control mechanisms
Type of control Description

Transcriptional Controls whether or not a gene is translated

(DNA to mRNA

Post- transcriptional Changes occur to mRNA molecules after

transcription ex. Introns removed and exons
spliced together

Translational Controls how often and how fast mRNA

molecules are translated

Post- translation Affect rate at which a protein b/c ‘active’ and

how long it remains functional

BIOTECHNOLOGY TOOLS
Science of biotechnology is based on recombining DNA of different organisms
Gene from one organism spliced into genome of another organism
Biotechnology relies on the three natural occurring classes of molecules that cut and splice
genes:
1. Restriction enzymes
➔ These are the molecular scissors that can cut double stranded DNA at specific
base pair sequences
➔ Each restriction enzyme recognizes a specific sequence of nucleotides known as
a recognition site
➔ Most recognition sites are 4-8 nucleotides in length and are usually
complementary Palindromic sequences
2. Methylase
➔ One of the roles of restriction enzymes in bacteria is to protect them from
infections by virus
Virus inject its own DNA into bacterium in order to reproduce however, restriction enzyme is
able to cut up viral DNA before it infects bacterial DNA
➔ Enzymes are able to aSite changes shape Dash blocking action of restriction
enzyme methyl side group (– CH3) to specific recognition site
➔ Sites changes shape – blocking action of restriction enzymes

3. DNA ligase
➔ DNA ligase is used to rejoin phosphodiester bonds that were broken by
restriction enzymes
➔ They can take a gene which has been ‘cut out’ of one organism and ‘splice’ it into
DNA of another
➔ DNA ligase works best when “sticky ends are available”
Bacteria have a large circular chromosome as well as many smaller circular structures called
plasmids. Plasmids are an important role in gene splicing
Gene splicing to produce Insulin
1. Human DNA is cut into fragments using restriction enzymes. Many fragments made, but
one will have insulin gene
2. Plasmids are cut with many restriction enzymes as in step one. Plasmids also contain
antibiotic resistance gene
(RESTRICTION ENZYMES MUST BE SPECIFICALLY CHOSEN TO CUT ON EITHER SIDES
OF INSULIN GENE)
- Using same restriction enzymes mean that plasmids and fragments have
complementary ends
- antibiotic resistance gene is important for a later process
3. Makes DNA fragment cut plasmids and DNA ligase, to produce recombinant DNA
plasmids
- Some recombinant plasmids will contain insulin gene and some will not
4. Through transformation, recommended plasmids enter bacterial cells
- As bacteria divide, billions of copies of recombinant plasmids are made
- Some of these bacteria will make insulin
 5. Process called hybridization used to identify bacteria colonies that produce insulin
Hybridization requires colonies to be antibiotic resistant
- These colonies will be isolated and given optimal conditions needed to producing
large quantities of insulin
GEL ELECTROPHORESIS
● Gel electrophoresis is a technique used to separate DNA fragments based on their size
● Sample of DNA extracted from tissue and exposed to one or more restriction enzyme –
cut DNA at specific recognizing sites
● Sample then placed into smaller plastic tube
● DNA sample will contain many different sized fragments
● Agarose gel used a filter to separate fragments based on size
● Technique can also be used to separate other organic molecules such as proteins
● DNA fragments placed in wells at one end of agarose gel
● Electric current placed across gel, causing fragments to move away from Wells
● Occurs because phosphate groups in DNA carries a negative charge which is attracted
to positive electrodes
● Smaller fragments move through gel more easily than larger fragments travel further
along in gel
● Fragments of same size move at same speed and group together
● Groups of fragments are still not visible, so stains are used to make DNA fragments
visible
● Commonly used stain is known as ethidium bromide - Insert itself along complementary
base pairs of DNA and Fluoresces under UV light
● Result is pattern of bands indicating groups of same sized fragments
● Length of these fragments can be determined by comparing position of each band to a
strand sample of DNA fragments

POLYMERASE CHAIN REACTION [PCR]

PCR – technique that allows scientist to make millions of copies of desired gene fragments in
hours
Developed by Dr. Kary Mullis in 1987
Won him Nobel prize in chemistry in 1992
Why is PCR technique important?
➔ PCR allows scientists to perform genetic tests on incredibly tiny amounts of tissue –
even a single cell.
➔ PCR can even be used on tissue that has been degraded by environment
➔ Used in medicine, genetics, biotechnology and forensics
How does PCR work?
1. DNA samples heated to 94°C to 96°C
- Heating breaks hydrogen bonds that hold double stranded do you need together
– complementary single strands
2. DNA primers added. Cool to 50°C to 65°C
- 2 primers (forward and reverse) is chosen to be complementary to opposite ends
of target region being copied
 3. Taq polymerase added. Heated it 72°C
- Tech polymerase comes bacterium Thermus aquaticus, found in Hot Spring -
enzyme does not denature at 72°C
- Taq polymerase joins at primers and move along strands adding complementary
bases pairs as it goes
- Result is two incomplete copies of DNA, each containing target region
CYCLE 1 COMPLETE
4. Repeat step 1-3 CYCLE 2
- Heated to 94°C to 96°C
- Add DNA primers; Cool to 50°C to 65°C
- Taq polymerase added; heated to 72°C
- After 2nd cycle result is 4 incomplete copies of DNA, each containing target region
5. Repeat CYCLE 3
- After the 3rd cycle there a total of 8 DNA copies, 2 of which are target fragments

DNA SEQUENCING
- process of determining exact order of nitrogen is bases is called dna sequencing
- In July 2000, two major American labs working on human genome project (HGP)
announced that 99% of our genes have been sequenced
- HGP used an automated process based on Sanger dideoxy method to sequence DNA
- Utilizes process of DNA replication
- Five components of need it for this technique:
1. A single strand of DNA template [to be sequenced]
2. Radioactively labelled DNA primer
3. DNA polymerase
4. Nucleoside triphosphate
5. Radioactively labelled dideoxy analogues
Sanger Dideoxy method
1. DNA to be sequenced is treated so that it becomes single-stranded (single stranded
DNA template)
2. Short, single-stranded radioactively labelled primers are added to the 3’ end of DNA
template
3. Thousands of identical copies of primed DNA template are placed in four reaction tubes
- Each contains DNA polymerase in all four nucleoside triphosphate [dATP, dGTP,
dCTT, dTTP]
- Small amount of a different radioactively labelled dideoxy analogues (either
dDATP, dDGTP, dDCTP, dDTTP)
4. Double stranded fragments that form in 4 reaction tubes are separated by length [using
gel electrophoresis]
5. Pattern of bonds formed on an autoradiogram can be used to decipher sequence of
DNA template
Dideoxy analogues
● Dideoxy analogues different from normal nucleoside triphosphates in that they are
missing an –OH group on 3’ carbon of their sugar unit
 ● As a result of missing, -OH group, DNA polymerase cannot join the next nucleotide to
dideoxy analogue
○ DNA replication stops when a dideoxy analogue is added
● dideoxy analogue acts as a chain Terminator whenever joins to DNA template
● Since small fraction of available nucleotides in each reaction tube dideoxy analogue,
different length of double stranded DNA will form before chain termination occurs
● Reaction vessels that contain dDGTP, would produce these fragments of double
stranded DNA
● Running results of all for reaction tubes on same gel results in a pattern that can be used
to determine sequence of DNA template
● Sequence can be read from an autoradiogram
● Sequence shown on autoradiogram is complement of DNA template
● It is easy to deduce sequence of template strand

GENETIC MUTATIONS
Mutation - Changes in DNA sequence caused by various mechanisms of spontaneous
mutations.
- occur during DNA replication
Enzymes quickly repair most errors.
Induced mutations are caused by mutagens
Mutagen- environmental agent that directly alters DNA within cell Radiation, chemicals, viruses
● Mutations in reproductive cells can be passed from one generation to next
● Mutations in somatic (body) cells do not affect future generation to next
● Inherited genetic disorders : cystic fibrosis, sickle cell anemia, Tay-sachs disease,
hemophilia, color-blindness, & achondroplasia
● Most inherited genetic diseases are recessive
● Person must inherit two copies of mutated gene to inherit disorder
● Two genetically similar adults are more likely to give a child two copies of defective gene
● Diseases caused by just one copy of defective gene (Huntington's disease) are rare
Types of Mutations
Small scale Mutation - mutations that involve changes to individual base pairs or small groups
of base pairs
Large Scale Mutation- mutations that involve changes in chromosomes and may involve
many genes
Small Scale Mutations
Point mutations: change in single nucleotide within a gene
- Mutation may be beneficial, harmful or neutral
- Three types : substitution, insertion/ deletion, inversion
- substitution- replacement of one base pair by another in a DNA sequence
- Insertion - addition of a base pair to a DNA sequence
- Deletion - removal of a base pair in a DNA sequence
- Inversion - two adjacent bases trade places
Insertions and deletions cause frameshifts
Frameshift- Shift in reading Frame resulting in multiple missense or nonsense mutation
Silent Mutations
● Change in one or more base pairs- results in new codon
● Mutated DNA sequence codes for same amino acid - resulting protein is not altered
● Does not affect functioning of gene - no change in phenotype
Missense Mutation
● Change in one or more base pairs - results in new codon
● Mutated DNA sequence codes for different amino acid - protein sequence & structure
affected
● Protein may be non functional or function differently
● Can be beneficial if it creates a desirable effect
Nonsense Mutations
● Change in one or more base pairs - results in a premature stop codon
● Polypeptide is cut short and most likely will be unable to function
Large Scale Mutations
● Involve multiple nucleotides, entire genes, or whole regions of Chromosomes
● Inversion - reversal in orientation of gene on chromosome
● Translocation movement of entire gene or sequences between chromosomes
● Gene amplification - multiple copies
Ex : Down Syndrome