Cellular and Molecular Biology

1. The central dogma of molecular biology is an explanation of the flow of genetic

information within a biological system. It is often stated as "DNA makes RNA and RNA
makes protein,"[1] although this is not its original meaning. The dogma classes these into 3
groups of 3: three general transfers (believed to occur normally in most cells), three special
transfers (known to occur, but only under specific conditions in case of some viruses or in
a laboratory), and three unknown transfers (believed never to occur). The general transfers
describe the normal flow of biological information: DNA can be copied to DNA (DNA
replication), DNA information can be copied into mRNA (transcription), and proteins can
be synthesized using the information in mRNA as a template (translation). The special
transfers describe: RNA being copied from RNA (RNA replication), DNA being
synthesised using an RNA template (reverse transcription), and proteins being synthesised
directly from a DNA template without the use of mRNA. The unknown transfers describe:
a protein being copied from a protein, synthesis of RNA using the primary structure of a
protein as a template, and DNA synthesis using the primary structure of a protein as a
template - these are not thought to naturally occur.
2. A. CRISPR (/ˈkrɪspər/) (clustered regularly interspaced short palindromic repeats) is
a family of DNA sequences found within the genomes of prokaryotic organisms such
as bacteria and archaea.[2] These sequences are derived from DNA fragments from viruses
that have previously infected the prokaryote and are used to detect and destroy DNA from
similar viruses during subsequent infections. Hence these sequences play a key role in the
antiviral defense system of prokaryotes. Cas9 (or "CRISPR-associated protein 9") is
an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands
of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with
CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be
used to edit genes within organisms.[3]This editing process has a wide variety of
applications including basic biological research, development of biotechnologyproducts,
and treatment of diseases.
B. Using their system, the molecular components of CRISPR are dried up and smeared
onto a strip of paper. Samples of bodily fluid (blood serum, urine or saliva) can be applied
to these strips in the field and, because they linked CRISPR components to fluorescent
particles, the amount of a specific virus in the sample can be quantified based on a visual
readout. A sample that glows bright green could indicate a life-threatening dengue virus
infection, for instance. The technology can also distinguish between bacterial species
(useful for diagnosing infection) and could even determine mutations specific to an
individual patient’s cancer (useful for personalized medicine).
C. Genetically modified organisms (GMOs) are organisms that have been altered using
genetic engineering methods. Although genetic engineering is a common and essential
practice in biotechnology, its specific use in crops is controversial. The key steps involved
in genetic engineering are identifying a trait of interest, isolating that trait, inserting that
trait into a desired organism, and then propagating that organism. Methods for genetic
manipulation have rapidly improved over the last century from simple selective breeding,
 to inserting genes from one organism into another, to more recent methods of directly
editing the genome.
3.A. Advantages with transposable elements:
When creating mutations there is only a single transposable present, and therefore the
number of second site mutations are low. This will save time when doing backcrossing.
B. Disadvantages with transposable elements:
Transposable elements are rather large and therefore not preferable to work with when
doing fine detail analysis (single nucleotides). Other disadvantages include merely
transient siRNA expression and low and variable transfection efficiency.
C. TEs are also a widely used tool for mutagenesis of most experimentally tractable
organisms. The Sleeping Beauty transposon system has been used extensively as an
insertional tag for identifying cancer genes.[50]
The Tc1/mariner-class of TEs Sleeping Beauty transposon system, awarded Molecule of
the Year in 2009,[51] is active in mammalian cells and is being investigated for use in
human gene therapy.[52][53][54] TEs are used for the reconstruction of phylogenies by the
means of presence/absence analyses.[55] transposons can act as biological mutagen in
bacteria.
D. A genome rearrangement is a major genomic mutation, usually driven by errors in cell
division following meiosis or mitosis. These large-scale changes to the structure
of chromosomes are almost always harmful and usually result in the death or sterility of
the developing organism, but in very rare cases, they provide a significant advantage. For
example, duplications of chromosomal segments can introduce an extra copy of a
beneficial gene, which allows one of the gene copies to evolve a modified function.
4. A. Classical breeding relies heavily on the naturally occurring plant life-cycle
and homologous recombination to generate genetic diversity and to eliminate undesirable
traits. It may also makes use of a variety of artificial laboratory procedures to overcome
obstacles to introduction of useful traits from wild species that do not usually exchange
genes with the domesticated line. These approaches include in vitro techniques such as
protoplast fusion, embryo rescue or mutagenesis (see below) to generate genetic alterations
and produce transgenic plants that would not exist in nature.
B. Molecular markers have become important tools in the hands of plant breeders for
enhancing the selection efficiency for various agronomic traits. Plant breeding has in fact
entered in an era of genomics. The isolation, cloning and moving of genes from diverse
biological sources into plant genomes holds promise to broaden the gene pool of crops and
tailor plant varieties for specific traits that determine yield, quality and resistance to biotic
and abiotic stresses. The molecular tools allow detection of specific DNA fragments
through successive generations, and thus confirm transmission of the selected traits and
incorporated genes. This book should help plant breeders to choose the appropriate
techniques suited to the needs of their respective breeding programs, and allow them to
integrate molecular tools based on gene cloning, gene maps, marker-assisted selection,
QTL mapping, and molecular cytogenetics
5. A. The three basic steps of DNA extraction are 1) lysis, 2) precipitation, and 3) purification.
Step 1: Lysis
In this step, the cell and the nucleus are broken open to release the DNA inside and there are two
ways to do this. First, mechanical disruption breaks open the cells. This can be done with a tissue
homogenizer (like a small blender), with a mortar and pestle, or by cutting the tissue into small
pieces. Mechanical disruption is particularly important when using plant cells because they have
a tough cell wall. Second, lysis uses detergents and enzymes such as Proteinase K to free the
DNA and dissolve cellular proteins.
Step 2: Precipitation
When you complete the lysis step, the DNA has been freed from the nucleus, but it is now mixed
with mashed up cell parts. Precipitation separates DNA from this cellular debris. First, Na+ ions
(sodium) neutralize the negative charges on the DNA molecules, which makes them more stable
and less water soluble. Next, alcohol (such as ethanol or isopropanol) is added and causes the
DNA to precipitate out of the aqueous solution because it is not soluble in alcohol.
Step 3: Purification
Now that DNA has been separated from the aqueous phase, it can be rinsed with alcohol to
remove any remaining unwanted material and cellular debris. At this point the purified DNA is
usually re-dissolved in water for easy handling and storage.

B. The first step in PCR, DNA denaturation, requires a high temperature, typically around 95
degrees Celsius. Denaturation causes the DNA to unzip and separate into single strands, exposing
the DNA bases to the rest of the PCR mixture.

The second step, primer annealing, must occur at a lower temperature than the denaturation step.
The PCR machine cools the solution to a temperature between 45 and 72 degrees Celsius. The
specific temperature for annealing depends on the primers. Primers are short pieces of previously
synthesized DNA that occur at the beginning and end of the DNA of interest. During primer
annealing, the primers bind to the appropriate parts of the DNA strand.

The third step, extension, occurs at 72 degrees Celsius. This step entails the extension of new
strands of DNA, starting with the primers.

After extension, the reaction is heated back to 95 degrees Celsius to start another cycle of PCR.
The number of strands of DNA after each cycle of PCR steps doubles, so the amount of DNA
produced is exponential. In this way, 20 to 35 cycles of PCR creates millions of strands of the
DNA of interest.

C. Some advantages are that it can create many copies of a minimal starting amount ie good for
criminal investigations if you find some hair you can use PCR to find out whose it is. Also
another advantage is PCR is speedy you can make tons of copies in hours.
Disadvantages are that with PCR cannot substitute for gene cloning in cells when large amounts
of a gene are desired. Also occasional errors during PCR replication impose limits on the number
of good copies that can be made.

D. Resequencing is often carried out by amplifying a specific region of the genome by PCR and then
sequencing the PCR fragment from both directions to generate a high-quality DNA sequence. Information
about the different strategies for resequencing are described in the Sequencing Chemistry Guide.

6. Storage of proteins for any length of time can pose stability problems. While working with
proteins in the lab, they should be kept on ice. Since proteins are generally more stable at colder
temperatures, maintenance at low temperatures even for short duration is
recommended. Typically, proteins are stored freeze-dried (lyophilized), frozen in appropriate
buffers, or refrigerated at 4°C. For short-term storage of proteins (hours to days), a standard
laboratory refrigerator at 4°C is satisfactory providing the buffer used to solvate the protein
provides all the necessary components necessary to stabilize the protein of interest. These
components can include reducing agents, hydrophobic additives, and protease inhibitors added to
buffers. Along with the use of gloves mentioned previously, protease inhibitors prevent
denaturation due to contamination from these lytic agents potentially present in the protein
source. Additionally, antibacterial agents such as sodium azide can be added to inhibit bacterial
growth. Care must be taken, however, since antibacterial agents and protease inhibitors represent
deliberate contamination of the sample. Proper controls must be evaluated to insure no deleterious
interaction with the protein of interest will occur.

7. A. A lot of reasons kill cells. Trauma, lack of oxygen, starvation... and there is also
programmed cell death (probably what you wanted). Apoptosis (a.k.a. programmed cell
death) is a cell's self-destruction (i.e. suicide). 1. Chromosomal DNA and nucleus break.
2. The cell shrinks. 3. It breaks into vesicles that can be "phagocytosed" (I'm not sure
this word exists) by other cells. If this didn't happen, you would not have your fingers
apart from each other (unless you cut the skin between them with a knife, I think).

B. Cells can die by apoptosis or necrosis. Apoptosis, programmed cell death, is usually
done in the name of the organism's survival. Necrosis is cell death by injury (acid,
freezing, burning, being torn open, etc). Cancer is the case of apoptosis failing... cancer
cells can't be programmed to die gracefully because some pathway in apoptosis has
become disrupted, so programmed cell death is a nice thing to have.

C. Ellagic acid is a naturally occurring polyphenolic constituent found in 46

different fruits and nuts such as grapes, pomegranate, red raspberry,
strawberry, blueberry, and walnuts. According to some sources,“[Ellagic acid]
prevents the binding of carcinogens to DNA and strengthens connective
tissue, which may keep cancer cells from spreading.” (1) Ellagic Acid is
claimed to have the ability to inhibit mutations within a cell's DNA.
Furthermore, it is considered to be a cancer inhibitor which has the ability to
cause apoptosis or normal cell death in cancer cells.
Working theories regarding the anti-cancer action of Ellagic Acid

 First, ellagic acid kills cancer cells by promoting cell death (apoptosis).
 Second, ellagic acid stops the growth of tumors.
 Third, ellagic acid causes G-arrest.
 Fourth, ellagic acid helps prevent cancer, birth defects, etc
 Fifth, ellagic acid seems to have anti-viral and anti-carcinogenic effects.
 Sixth, ellagic acid (i.e. from ellagitannins), is combined with glucose,
making it easier to gain entry into cancer cells.

8.