Molecular Biology-Lecture 1

5 questions
1. What is a gene? *You have to know this*
A hereditary piece of DNA information that produces a functional RNA
molecule. That RNA is translated into protein, but a lot are not.
2. What is an allele?
A variant form of a gene.
3. What is transcription?
Enzyme that preforms transcription is RNA polymerase. Uses DNA as a
template to make RNA. All genes are transcribed by RNA pol, no exceptions.
4. What is a single nucleotide polymorphism (SNP)?
A difference in sequence of a nucleotide in a population. These SNPs make us
who we are.
5. Are mutations always bad? If not, give an example.
No. Example, sickle cell anemia. If youre a hetorozygote you are more
resistant to malaria. If youre a homozygote for the mutation then you get
sickle cell anemia.

Your Genome:
How much DNA does each of your cells contain?
1 metre – 3 billion nucleotides (for a haploid) Inside the nuclei of every single one of
your cells. 6 billion nucleotides makes us who we are. Would take 57 years to read
your genome.

Human Genome Project:

 Completed April 2013
 Entire nucleotide sequence of our chromosomes
 20-25,000 genes
 Cost 3 billion dollars
 Human genome: quality assessment of the human genome sequence

Personalized medicine:
 Your individual genome sequence
 Determine susceptibility to disease
 Which medications you will respond to or react to (Thousands of deaths per
year due to adverse side effects of prescription drugs)
 Will be used in doctor’s offices in the next 10 years
 Important to understand what this information means

What can you get today for $99?

 Not complete genomic sequence
 Sequences of thousands of genetic markers >1000000 base pairs (SNPs)
 23andMe (company)
  Using genomic information for disease discovery treatment and drug
effectiveness (pharmacogenomics)

Ethical questions:
 What to do with this information without treatment for some diseases?
 Psychology impacts on individuals and their disease risks
 Impacts on insurance policies and job security
 Getting significant other to get tested before making commitments

Other Genome Projects

How many of your own cells in your body? 20-30 trillion
How many total cells in/on your body? 150 trillion

Human Microbiome project

The Human Microbiome is the collection of all the microorganisms living in
association with the human body. These communities consist of a variety of
microorganisms including eukaryotes, archaea, bacteria and viruses.

Bacterial diversity on the human body

 Very few bacterial species can be cultured in lab (<10%)
 New DNA sequencing technologies can now identify many unknown bacterial
special without culturing

Genomes and Evolution

Chimps and humans
 2.7% of genomes are different
 some genes have been mutated or duplicated
 FoxP2: differs by two amino acids, involved in speech development

History of Molecular Biology

Field has changed immensely in the past years, continues to change.
Has only been around for about 100 years

Chromosome is a single double stranded molecules containing hundreds of

nucleotides that contains genes
Chromosomes contain thousands of thousands of genes along its length
Not all of them are turned on at the same time, some are turned off

What is the definition of a gene? ***on the exam***

Heritable sequence of DNA that encodes a functional RNA molecule; in protein

coding genes, the RNA in turn codes for protein
-Some genes encode RNA that is not translated into protein e.g, rRNA, tRNA,
-miRNA- found everywhere, critical roles in regulating translation in protein coding
genes. Produce a transcript which is used to bind to other target RNAs. Many
cancers are the result of miRNA expressions that are not proper. Major role in
proper regulation of gene expression .
-PiRNA-produced in some tissues at higher levels than other especially in testes.
Play a role in sperm development. Very numerous
-siRNA

(last 3 play role in regulating gene expression)

-RNA can be reversed transcribed back into DNA (only in viruses- have an RNA
genome that have an enzyme, reverse transcriptase)
DNA Replication: DNA polymerase dsDNA dsDNA
Transcription: RNA polymerase dsDNAssRNA
Reverse Transcriptase: Reverse transcriptase ssRNA dsDNA

DNA is the genetic material (has coding capacity) and can transform cells (change
cells)
I928: Griffith experiment using Pneumococcus
Proved that virulence can be passed between cells via DNA transformation
Fig. 1.3
-Avirulent (nonlethal) R type bacteria could be converted to lethal S (smooth)
bacteria when cells were mixed together.
-All mice became lethal when R and S were mixed together.
-Something in S converted something in R strain to make it virulent
-We know now that DNA is the transforming principle
-DNA is a very stable compound; RNA is not very stable (will break down to heat
etc)
-They took the dead mice and recovered the bacteria that killed it and extracted the
DNA from those cells.

DNA is Genetic Material of Viruses

Hersey and Chase experiment (1952)
-used radioactive compounds to tag DNA (32 P) and protein (35 S) of viruses (phage
T2)
-infected bacteria and recovered viral progeny and measured radioactivity amounts
-virus particle (sulphur part?) stays out, only DNA goes in
-New viruses had high levels of P32 but not S35

Fig.1.6
DNA can be introduced into all cells
-DNA can be transformed into all cells
-When DNA is put into eurkaryotic cells it is termed transfection which is the same
as transformation into bacteria
-DNA transforms at LOW efficiency
-Need a way to select for cells that have taken up DNA
-Selectable markers
-Antibiotic resistance in bacteria

DNA structure
- DNA is a double helix with an average of 10 bp/turn
- Overwound DNA has more bp/turn and under wound has less
- The two strands of helix run antiparallel
C has triple bond to G
T has double bond to A

What are the 3 building blocks of nucleic acids

- Nitrogenous base
- Phosphate
- Sugar

DNA supercoiling
-DNA can coil around its axis if it is closed with no free ends (e.g. plasmid- closed
circular DNA)
-Supercoiling affects the structure of DNA: A closed DNA can be a circular DNA
molecule or a linear molecule where both ends are anchored in a protein structure
-Positive supercoiling: Twisting of the DNA helix in the same direction as the two
strands; causes increase in the number of bases/turns. Increases supercoiling
capacity of DNA (difficult to pull apart)
-Negative supercoiling: Twisting of the DNA helix in the opposite direction as the
two strands; causes decrease in the number of bases/turns (easy to pull apart) bc
helical structure is being disrupted, and doesn’t take much to peel them apart.
Causes strand separation (denaturation).

Why is this important?

-DNA Replication
-Transcription

Transcription and Replication require DNA strand separation and special enzymes
DNA strands have to separate during replication transcription and recombination
How does the torsional stress get relieved?
-A nick (break) is generated in one of the strands which allows it to rotate to relieve
tension
-Nick is resealed (ligated) after its rotated around the intact strand
-Many proteins (enzymes) are involved in this process
Topoisomerases
Can relax OR create supercoils in DNA
Break bonds in DNA
Type 1- single strand breaks
Type 2- double strand breaks

Gyrases can also affect supercoiling

Enzymes involved in DNA degradation

- Deoxyribonucleases (DNases) degrade DNA
- Ribonucleases (RNases) degrade RNA
- Endonucleases: break bonds within nucleic acid
- Exonucleases: break bonds at ends of nucleic acid (5’ OR 3’ ends)

DNA replication theories

1.Semiconservative
2.Conservative*
3.Dispersive*

DNA replication is Semiconservative

DNA duplex serves as a template for the production of a new strand
Meselson-Stahl Expt (1958) labeled DNA with heavy isotope Nitrogen and watched
generation of new strands.
Measured the weight of DNA using density centrifugation

Nucleic Acid Hybridization

All nucleic acids can hybridize (form H bonds) with each other using
complementary sequences. As long as the nucleotides pair up.
DNA-DNA, RNA-DNA, RNA-RNA
Intra- or intermolecular bonding. Most nucleic acids are long, so if you have a single
RNA molecules that has a bunch of C’s and G’s, they can fold back and create a
double stranded RNA. (can happen with DNA too)
As long as there is homology is the sequence, they can base pair.
Heating can lead to strand separation or denaturation of DNA duplex (‘melting’ of
DNA)
Melting point (TM) is the midpoint of the range over which DNA strands separate
TM is dependent on the length of DNA and G-C content
Typically 40% GC DNA has TM of 87 Celsius
Both DNA and RNA may form duplexes (figure 1.22)
-depends on hybridization of nucleic acid molcules

DNA can be denatured and renatured(fig1.23)

Apply heat break H-bonds, bring temperature down, DNA will re-anneal
Renaturation of nucleic acid strands
Any two strands with complementary sequences can anneal, or renature (or
hybridize)
Ability of two strands to hybridize is directly related to complemtarity
Can still get hybridization with sequences that are not 100% homologous (imperfect
duplex)

Many molecular biology techniques rely on hybridization

Allows for detection of specific DNA or RNA sequences
Southern blotting-DNA detection
Northern blotting- RNA detection

The filter assay measures complementarity (fig 1.24)

Denature DNA and put it on filter
Take probe (mouse) denature in solution
Put both strands in solution and measure DNA bound to filter
DNA is usually radioactively labeled so detection can be performed using X-Ray film
(autoradiography)
Pull out hybrid and get it sequenced, clone it.
Provided foundation for molecular bio
Both probe and target have to be single-stranded (DNA and/or RNA)

DNA mutations
Questions about mutations and populations:
What has to occur in the population for beneficial mutations to be successful and
become heritable?
Fitness has to improve
Reproduction
Selective pressures to isolate and give selective advantages to individuals in the
population so they may survive

Why are serious mutations not generally observed in the population?

Really serious mutations don’t make it through the developmental process, aren’t
born. If they are born, they don’t reproduce because mutation is too severe.
What percentage of fertilized eggs make it till birth? 20%

All mutations are changes in the genomic DNA sequence

Not all mutations are determined or affect phenotype of organisms; some may be
beneficial and lead to evolution
Spontaneous mutation: result from random cellular events (e.g. mistakes in DNA
replication); extremely rare
We have mechanisms to fix these mistakes

Mutagens: chemicals or compounds that increase background level of mutation-

induce mutations
- normally act directly to change DNA base pairs
DNA mutation and disease:
If mutations occur in germ line cells- passed on to next generation
Estimated over 100 genetic diseases that result from mutation in single genes
Some mutations occur spontaneously in an individual- didn’t inherit from parents

Is cystic fibrosis hereditary?

Yes, 28 different mutations in the CFTR gene are known to cause the disease

Is liver cancer hereditary?

In >95% of cases = NO
- mutations can happen in somatic cells- not passed on

Recent study found that >60% of cancers are due to what?

Random mutations unaffected by lifestyle choices (bad luck)

Mutations can be detected and repaired in cells

Mutation rate of genes is dependent on genome size and selective pressure applied
on organism

Genome sizes vary dramatically between species!!

-Has major impact on evolution of species

(plants have the biggest genomes)

Beneficial mutations:
-Some mutations provide benefit for survival
Example: CCR5 gene  )- protects you against HIV  encodes chemokine receptor
5
(CCR5delta32 mutationnon functional
Found that patients homozygous for CCR5delta32 were resistant to HIV infection
(mostly of European origin)
-HIV uses receptor to infect white blood cells
Why does this mutation exist in the population?
Bubonic plague (14th century Europe)  caused by bacteria
CCR5delta32 leads to plague resistance ???

CCR5delta32 mutation is a deletion of 32 base pairs in the gene that inactivates it

and doesn’t allow HIV to bind to T-cell

Disease causing genes with benefits depending on gene allele copy number (homo
vs. heterozygous)

Cystic Fibrosis
1 in 50 caucasion individuals carry the recessive allele
homozygous recessive alleles for CFTR mutation
Causes mucus to form in lungs and intestines – once you have mucus, bacteria can
get in and create own environments, continue to grow, hard to get rid of because
protected by mucus

Heterozygous advantage
- one copy of recessive allele provides resistance to diarrhea (caused by
cholera) and resistance to tuberculosis
- tuberculosis responsible for 20% of European deaths between 1600-1900
- in heterozygous state could provide protection to individual, homozygous
disease

Types of Mutations
1. Point mutations: Change in single base pair
Transition: Replaces pyrimidine with pyrimidine and vice versa
Transversion: Purine replaced by pyrimidine and vice versa
- Mutations can be induced by chemical modifications of bases or
incorporations of base analogs into DNA

Nitrous acid deaminates cytosine to uracil

Changes structure of base, not sugar. C converted to U which is paired with G. RNA
would eventually base pair with A. Eventually over time that C-G base pair will turn
into a T-A. Some cells will contain the C-G pair some will contain the other.

Opposite: Base analogs

Should know that it acts differently in terms of mutagens.
Analogs look like a base, not exactly the same
At high enough doses, this will kill the cell totally messes up point mutations all
over, cell cant handle it.
Uses:
-explains why cigarette smoke induces lung cancer.
-Chemotherapy (anti-metabolites): cancer cells take up analogs and block division
and DNA replication. Leads to cell death.
Purine Analogs: mercaptoguanine
Pyrimidine Analogs: fluorouracil
- Eventually, cancer cells die but also normal cells can die too.
- Causes side effects of chemotherapy (because kills normal cells that have
high replicative capacity- hair, skin, epithelial)
- Also targets auto-immune cells

2. Insertion: Addition of more than on bp into genome

3. Deletion: Removal of base pairs from genome
4.Transposons: Sequence of DNA that can duplicate and insert into new location in
genome. How much of our genome is composed of transposons? 45%.

Mutation Reversion
Just because there is a mutation, doesn’t mean it can’t correct it.
Point mutations and insertions can be reverted back to wild type sequence;
deletions cannot.
Forward mutations: inactivate a gene. You create an insertion or point mutation
which leads to the inactivation. (wild-type to inactive)
Backward mutations: restores inactive gene to wild-type. Inactive gene is now
activated.

Exact base reversal is true reversion

Base reversal somewhere else in gene compensates for initial mutation: Second site
reversion. Restores gene function.

What is a genome?
The collection of DNA in an organism.

Genome difference
All cells have dsDNA genomes, some viruses also have dsDNA, ssDNA, or RNA
genomes
All genomes code for the production of all the protein for a cell in an organism.
All the collection of proteins in an organism provides all the necessary functions for
survival of the organism
What is difference between genome and proteome? Why?
One is DNA one is protein.
A particular genome gives rise to a particular proteome.
Every somatic cell in your body should have same genomes
Proteomes do not have same genomes
The thing that is different is expression: the types of genes that are turned on, the
types of proteins that are made.
Proteomes are going to be roughly the same
The brain proteome and the skin proteome are different.
But two different brain proteomes will the similar.
If there are 20,000 genes in the human genome, how many proteins are there?
Estimated that our bodies produce 125, 000 proteins for 20,000 genes.
How does this happen? Alternative splicing.
One gene can give rise to multiple RNA’s due to this.
Almost every one of our genes can make at least 2 different protein copies.

Viroids: unique type of plant infectious agent that doesn’t contain any protein
associated with it (ssRNA with high intermolecular complementarity)  viroids do
not encode any protein
How are they pathogenic??
Inhibit gene translation
Ribozymes can cleave RNA. It can also bind to a target mRNA and prevent
translation and cause that plant problems, thus causes disease

-May act as a ribozyme or inhibit translation or normal genes in plants

some are transcribed by RNA poll1 into rolling circle RNA
Example: potato spindle tuber viroid (PSTV), cadang cadang viroid (242 base
ssRNA)  infects coconut plants. So they play a role in agriculture

how does it make more of itself ?  it uses the host to make more of itself

Prions:
- Infectious agents that do not contain DNA or RNA
- Don’t have a genome at all. No DNA.
- Infectious proteins.
- These proteins can be cause of mad cow disease as well as some forms of
disease
- Based on particular gene that all animals have (all mammals, even yeast have
it)
- 28kDa hydrophobic glycoprotein encoded by normal gene in brain
(mammals)
- Two Forms:
1. PrPc: normal and degraded by proteases in cell
2. PrPsc: resistant to degradation- cant degrade

PrPc is converted to PrPsc to cause disease.

- When it sees a protein similar to itself.
- Causes neural death and degradation, then you die.

Chapter 2: Genes code for Proteins

A chromosome contains a very long stretch of DNA that contains many genes
Each gene is part of a continuous sequence of DNA.
We all have 23 pairs of chromosomes. Each chromosome is one long strand of
double stranded DNA molecule which contains many genes.
If you blow up one gene, there’s a beginning and end, promoters, etc.
Fundamentally a stretch of ACTG’s.

What controls genetic variability? (changes in sequence, SNPS)

99.5% of our DNA sequences are the same (between humans)
Alleles: alternate forms of a gene (may be many for a single gene)
You need to generate genetic variability all the time within a population.
Locus: region on the chromosome where a gene is located
Different sequences at the locus, will generate different alleles.
-genes on a specific chromosome are said to be linked
- they do not assort independently during meiosis as do genes that are on different
chromosomes.
-Recombination can occur between homologous chromosomes during meiosis
(crossing over).
-causes genetic variation

What other ways can you get genetic variation?

For every gene you have a paternal allele and a maternal allele. For every gene we
are diploid. FALSE?
Dr. Stephen Sherer (Windsor, ON)
Studies variability in genome how it affects humans and causes disease
-You get one maternal and one paternal copy for each gene ?????
in 2006- new discovery
“Global variation in copy number in the human genome”
- Compared genome sequences from 270 people from 4 populations
(descendants from Europe, Asia, Africa)
- Found tremendous variation in human genome depending on where you’re
from

They isolated stretches of the genome that were found to be variable in the human
genome. Started to line up all sequences, and they found huge gaps between
populations. These gaps are called:
CNVs: copy number variations (Regions of the genome >1000 bps)
-Insertions, duplications, and deletions
-1446 CNVs account for ~12% of genome (total of 260 megabases)
image- all the chromosomes, blue lines show where a CNV exists, red lines are
frequency of it between populations. Theyre everywhere, all the way through the
genome. In some regions, big chunks that are gone have genes in there.
Different possibilities in population:
1 from mother, 3 from father
or
2 from mother, none from father

4 copies is going to cause problems vs missing paternal.

4 copies has to deal with levels of expression, twice as much gene product, has
affects on cell.

Some don’t contain any genes, some do.

What does all this mean?
-For some genes we may inherit more or less genes from one parent (genomic copy
number)
-not the same amount from each parent
-more or less than 2
-implications for disease traits where increase or decrease in gene expression may
be a cause, regulation of all of these genes, when things get out of balance you can
get diseases
- genomically sequenced individuals who have autism. Compared genomes and
isolated 100 different genes that played in development of role of autism.
- autism is extremely complex.

1000 genome project

-sequencing genomes of 1000 people from different populations around the world
to compare variability.

CNV number plays a role

Example, Charcot- marie- tooth type 1A gene
Depending on how much you have, leads to different diseases

CMT disease
-most common nerve related disorder
Leads to myelin sheath damage in peripheral nerves
Diagnosed in mid-childhoos and progressively gets worse
Caused by mutation in PMP22 gene
Another 43 genes involved in other CMT phenotypes
You can get the disease without the mutation – all has to do with copy number – if
you have too many in your genome you can get the disease.

PMP22 gene: copy number and impact on disease

*figure
difficult to diagnose

copy number variation for specific genes vary between population

why?

Human evolution and copy number variation:

Can copy number variation explain some human phenotypes?
Some cases we have clear evidence that increasing copy number has to do with
evolution. Has to be something adaptable, selective.
Diet  high vs low starch  can select for high amylase gene (AMY1) copy number.
-specific genes that break that starch down
-more than 2 copies of AMY1
-looked at populations with clear separations in diet choices, looked at genome
sequences, how many AMY1. Populations with high starch diets had higher copy
number, tribes with low starch content had not evolved additional copies of that
gene. *figure
therefore high starch diets = high amylase copy number

RECAP
If sequences of gene X are different, they are different alleles of gene X (also SNPs)
If gene X is duplicated, creates a new CNV

One gene= one enzyme hypothesis(1909/1941)

Some enzymes are produced by >1 gene
One gene=single polypeptide (1962)

Some genes only encode RNA

Gene is a heritable sequence of DNA that encodes a functional RNA molecules
(2015)
Most mutations that affect gene function are recessive
Get absence or reduction in protein function

Recessive alleles do not product active protein figure 2.2

Types of mutations:
Null mutant: eliminates function of a gene (no protein produces, no rna transcribed)
- Sometimes these are lethal mutations (organism dies)
Silent mutation: usually point mutations that change the DNA sequence but not the
protein sequence
Neutral mutations: changes in the DNA sequence resulting in changed amino acid
sequence that does not affect protein activity.
Leaky mutations: may affect protein function but not enough to change phenotype
Loss of function mutations: lead to loss in gene function (but not usually complete
loss)
Gain of function mutation: mutations that cause new function for a protein (rare but
usually dominant)

Multiple Alleles:
Usually multiple mutations in a gene results in many different alleles
Can have a heterozygote that contains two mutant alleles
Eye colour in drosophila (fly)
W+= wild type normal (red)
Wi= white eye (no pigment)
W+Wi= heterozygote is red (dominant)
WiWi= homozygote is white (recessive)

Some genes have more than one wilde type allele

Polymorphism: many alleles with no one allele being considered as wild-type
(usually >1% of population)
e.g.: ABO blood group locus on chromosome 9

A alleles: produce glycosyltransferase that uses a cofactor (UDP-N-acetylgalactose)

hich produces A antigen
B allele: produces glycosyltranserfase that uses a cofactor (UDP- galactose) which
produces B-antigen
O allele: mutation (deletion) in exon 6 which produces no enzyme

What is the structure of a gene on the chromosome?

Composed of AGCT.
How do we know how many genes are in our genome?
Look for specific sequences that are found in the promoters of genes.
Not just TATA box, a whole bunch of other ones that have a specific sequence.
Can we predict the function of a gene by its DNA sequence?

Structure of a gene
6 billion base pairs, only a small % considered genes.
How do we know where genes are in the genome and what they actually make

All genes have switches that turn the dna on and off. The expression is different if
youre in liver, brain, etc. Directed by sequences found in promoter.
Promoters require binding of proteins produced from different genes to activate
transcription of gene X.
If everything is done at appropriate time then signal is given to start transcription.

Transcription:
One strand of DNA serves as template for production of mRA.

One strand is the coding strand, the other is the template strand. SO only one strand
of the double helix acts as the template strand.

Complete mRNA contains 5’ untranslated region (UTR) called leader.

mRA also contains 3’ UTR called trailer
fig.2.14 * yellow part is the codons
ribosome binds to 5’ end and stops at stop codon. Its that length of RNA that defines
the gene.
The promoter has additional sequences that binds to specific proteins that turn on
that gene or turn it off.
Fine molecule only has exons (mature RNA) because it spliced out introns.

Bacteria:
Bacterial transcription and translation takes place in the cell at the same time. Why?
There is no nucleus. Ribosome, RNA pol, all in the same compartment. Bacteria
doesn’t have introns, so splicing doesn’t have to occur.

Eukaryotes:
Transcription takes place in the nucleus and translation in the cytoplasm
mRNA is transported from nucleus to cytoplasm
Protein production: Translation
Only one strand of DNA serves to prduce RNA template via transcription
Genetic code is read in groups of 3
Gene includes series of codons that are read sequentially from starting point to a
termination point in 5’-3’ direction
Codons are non-overlapping

Gene reading frames:

-3 possible ways of reading an RNA sequence

Open reading frame (ORF):

 Reading frame used to produce the protein from a particular gene
 ORFs start with an AUG initiator codon and end with a stop codon (UAA, UAG,
UGA)
 Any mutations within the ORFs can alter accurate protein translation
(blocking reading frame)

URF: unidentified reading frame

- An ORF for which no protein has been identified
e.g. FLJI0560

Gene and protein sequences are collinear:

Sequence of nucleotides is same as sequence of amino acids:
3N bp= N amino acids

Translational mutations:
Insertions and deletions into the DNA can lead to amino acid changes in the protein

Frameshift mutation: Changes the reading frame of the codons, usually causes
severe effects

Acridines: chemicals that induce structural distortion in DNA-leads to frameshift

mutations

If insertions or deletions are in mutiples of 3 nucleotides, less severe consequences

result.

Cis vs. trans

Cis acting acts on ITS OWN molecule (usually DNA)
Found within DNA sequences, within its own gene

Trans-acting can function on OTHER molecules (protein or RNA) .. things are

moving around (Transit)
All gene products (protein and RNA) can act in trans
Diffusible in the cell and can act on other molecules
Binding sites on the same DNA molecule are cis-acting
A gene can be inactivated by a mutation in a control region or in the coding region of
a gene
These mutations cannot be distinguished genetically
A gene can be inactived by a mutation in the cis-cting dna element that controls gene
expression or in another gene that acts in the trans to regulate the gene

Chapter 3: Genetic Engineering Technology

Work on bacteria and viruses in the 1970s and 1980s lef to development of
recombinant DNA technology and genetic engineering

Foreign DNA could be manipulated and transferred into other organisms (e.g human
DNA functions in bacteria)
Allowed for human genes to be isolated and the proteins produced in the bacteria
e.g human insulin approved in 1982 for diabetes treatment (produced in bacteria)
Epogen- human erythropoietin (produced in mammalian cell culture by amgen)

Best selling recombinant DNA drugs (biologies)

Recombinant DNA Technologies

Cloning Vector –circular dsDNA plasmid that is used as a vehicle to transfer DNA
into organisms
Foreign DNA can be inserted into vector using restriction endonucleases and DNA
ligase.
All vectors have selectable marker and origin of replication for appropriate host
(bacteria, yeast)
Shuttle vectors: DNA plasmids that can be transferred between different types of
organisms
Reporter genes are used to measure the expression levels of promoters and whether
DNA was taken up by the cell (B-galactosidase (LacZ), luciferase, GFP)

Restriction endonucleases can leave stick or blunt ends.

Other types of DNA transformation:

Viral: virus vector to infect bacteria and inject DNA. 100% efficient. Will affect every
type of cell.
Liposomes: DNA is coated with lipid membrane and DNA/lipid complex fuses with
plasma membrane (used for mammalian cells). Theyre just lipid molecules, that
lipid forms a lip bilayer, that encapsulates the dna inside of it. When that liposome
hits the cell membrane, the membranes fuse and the DNA goes inside the cell
Microinjection: DNA is injected into cells (usually fertilized eggs) – physically
injected. Recombined into the genome.
Biolistics (nanospheres): DNA is coated on a bead of gold or tungsten and fired into
cell using gene gun (plant cells).

Types of DNA vectors:

Plasmid: used for bacteria, yast, mammals, and plants
Phage: used for bacteria
Artificial chromosomes: bacterial (BAC) and yeast (YAC)
YAC’s have centromere and telomere
-Type of vector chosen depends on size of DNA fragment
-YACs are used in genome sequencing projects

Genome sequencing problems (look at slide)

DNA sequencing technology (Sanger method)

Enzyme that does this reaction- DNA pol

How does 23andME sequence SNPs?

-get individuals genomic DNA from cheek cells in saliva
-use sequencing system developed by Illumina Corp.
-use infinium HD assay
-Requires slide array with 1.2 million beads (3mm), each containing one 50-mer
primer containing one SNP

Illumina stock price

~500% increase over 3 years

Illumina/23andMe SNP sequencing

SNP – rs11868035
(A or G)
Chrom #17

What about whole genome sequencing?

Nanopore DNA sequencing
Types of nanopore sequencers:
1. Biological: uses pore-forming protein in a membrane such as a lipid bilayer. Very
small. Need them to survive. We have many pores that allow ions and small ions to
go in and out of the cell.

Example: channel pore proteins (alpha-hemolysin).

- heptameric protein pore with an inner diameter of 1nm. You have to be
smaller than 1nm to get through these pores. 7 subunits that form the
structure of a nanopore. Pore gives signal when interacting with
DNA/RNA /protein. They take this protein and they put it in a platform that
can measure electrical charges differences as molecules are fed through this
protein. Artificial nanopores created by proteins in a matrix that allows them
 to process DNA through the pore, as it goes through it changes the charge as
it is read across the membrane.

3. Solid state: fabricated from synthetic materials. No biological proteins involved.

Complete chem and physics.
Example: Graphene: pure carbon similar to graphite (but only one atom thick).
- Nobel prize for physics (2010)
- Incredibly strong, 100x stronger than steel
- Conducts electricity much better than copper wire

How does nanopore sequencing work:

- Single stranded DNA is fed through a hole (pore) in a grapheme
nanosequencer which has electrical sensor built into pore.
- Electrical signal excited by each nucleotide as it enters pore is different.
- Differences in signal can be analyzed by computer and converted into DNA
sequencing information

Advantages of nanopore sequencing

1. Fast (250,000 bp/sec/pore)
2. DNA is not degraded. Can re-sequence.
3. Cheap
Future Advancements:
1. Disposable sequencers ($50)
2. 8000 pores/device
How long would it take to sequence the human genome with 8000 pore
nanosequencer?

DNA sequencing technologies

Dideoxy termination
Advantage: Reliable, inexpensive
Disadvantage: short sequences, DNA is degraded

Illumina
Advantages: fast, cheap, scalable
Disadvantages: gives only SNP data, DNA cannot be reused.

DNA nanopore
Advantages: fast, cheap, high throughput, DNA not degraded
Disadvantages: error rate is high

Microarray (DNA chip) technology

Every gene from organism is spotted onto glass slide (tiling array)
mRNA is isolated from different tissues and cDNA is made with fluorescently labeled
nucleotides.
-Only single stranded DNA
- Labeled cDNA from different tissues is used to probe slide (example: cancer (red)
vs. normal (green).
- Differences in gene expression between tissues can be measured by quantifying
different colour intensities.

Nucleic Acid Detection

- To detect nucleic acids, they have to be denatured.
- Nucleic acid prove of interest has to be labeled (Radioactivity, fluorescently)
- Nucleic acid is usually transferred from a gel to a solid support (filter),
probed, washed and then exposed
- Or probing can be done in cell (in situ hybridization)

Gene detection using hybridization

-if you have a gene that you want to identify its location on a chromosome you
use in situ hybridization
can take intact cells or mitotic chromosome spreads and add denatured
fluorescently labeled DNA probe.

How are proteins visualized?

Chemiliminescence
Chemical generation of light

Chapter 4: The interrupted gene

Eukaryotic gene structure
-pre-mRNA contains exons and introns.
-mRNA only contains exons
-introns removed in the nucleus before transport into cytoplasm
-RNA splicing involves many enzymes
-Both pre-mRNA and mRNA contain 5’ and 3’ UTRs.
-exon order does not change between DNA and pre-mRNA

Humans: ~20,000 genes.

Differential splicing is critical machinery that has allowed us to evolve.
Now estimated 90-95% of our coding genes are differentially spliced.
One gene has the potential to produce 38 000 proteins. That gene is involved in
generation of down syndrome.

Exons are very small. Smallest part of gene. Introns are big.
Exons are building blocks of genes, give rise to different structure. Mixing and
matching.

What you can get is the elimination of exons

At some point in the nucleus, splicing occurs,
introns are removed
exons are building blocks to create a certain type of protein
whats bigger? Exons or introns? Introns.
How can you determine where the introns are?
There are specific sites/sequences that the splicing machinery sees as a
splice site, it can relegate the RNA
Before the genome sequence, you could compare the mRNA with the DNA
you can align genomic copy of the gene, and the mRNA and you can see where the
introns are
itron position is convered:
now people can look at specific genes that are conserved in the animal
kingdom, etc. intron positions are highly conserved. The exons are broken up in the
same way in all species
they perform a very important function in the survival of that species
introns are found in the same positions relative to the exons, however the length of
introns can vary widely
what does change is the size of the genomic gene, it is made up of intron sequences

Intron position is conserved: WHY?

MICROEXONS
-major role: development of the brain
-present in every cell in our body, every somatic cell has them. Only used in nervous
system. Only time they are ever spliced.

Figure:
If splicing factor is there in the cells, splicing in between 2 exons in mRNA
translated.
Blue and gold have to interact to get proper neuronal cells ?
Non-neuronal -- > leads to autism (abnormal neuronal)