solutions manual for biotechnology fundamentals … · q3. explain mendelian’s genetics. answer:...
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SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
SOLUTIONS
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
7
Q4 True
Q5 Human insulin
Q6 1991
Q7 False
Q8 Restriction endonucleases
Q9 True
Q10 Golden rice
Section C: Critical Thinking
Answers are not provided in this section as the students are encouraged to write their
own viewpoints.
CHAPTER 2 GENES & GENOMICS
Section A: Descriptive Type
Q1. What is the cell theory?
Answer: The idea of the cell theory is coined by a Frenchman, H.J. Dutrochet, and the
credit for formulating the cell theory is given to a German botanist, M.I. Schleiden and a
German zoologist, T. Schwann, who clearly outlines the basic features of the theory in
1839. Moreover, in the year 1858, R. Virchow extended the cell theory and suggested
that all living cells arise from pre-existing living cells. To prove Virchow hypothesis,
Louis Pasteur performed experiments suggested that living things are composed of cells
and all living cells are arise from pre-existing cells. There is an exception that does not fit
into cell theory, such as viruses which may be defined as an infectious subcellular and
ultramicroscopic organism. The viruses are simple as they lack the internal organization
which is the main characteristics of a living cell and due to this unique characteristics,
viruses, mycoplasma, viroids, and prions do not easily fit in the definition of a cell
theory. In addition, there are other organisms in protozoa and algae, which also don’t fit
in the definition of cell theory.
Q2. Describe the characteristics of a prokaryotic cell.
Answer: The prokaryote cell is simpler than a eukaryote cell, lacking a nucleus and most
of the other organelles of eukaryotes. There are two kinds of prokaryotes: bacteria and
archaea; they share a similar overall structure. A prokaryotic cell has three architectural
regions: (a) on the outside, flagella and pili project from the cell's surface. These are
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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structures (not present in all prokaryotes) made of proteins that facilitate movement and
communication between cells; (b) enclosing the cell is the cell envelope generally
consisting of a cell wall covering a plasma membrane though some bacteria also have a
further covering layer called a capsule. The envelope gives rigidity to the cell and
separates the interior of the cell from its environment, serving as a protective filter.
Though most prokaryotes have a cell wall, there are exceptions, such as Mycoplasma
(bacteria) and Thermoplasma (archaea)). The cell wall consists of peptidoglycan in
bacteria, and acts as an additional barrier against exterior forces. It also prevents the cell
from expanding and finally bursting (cytolysis) from osmotic pressure against a
hypotonic environment. Some eukaryote cells (plant cells and fungi cells) also have a cell
wall; (c) inside the cell is the cytoplasmic region that contain the cell genome (DNA) and
ribosomes and various sorts of inclusions. A prokaryotic chromosome is usually a
circular in a shape with an exception is that of the bacterium Borrelia burgdorferi, which
causes Lyme disease, DNA is linear in shape. One of the distinct features of prokaryote is
the absence of nucleus in the cytoplasm; DNA is usually condensed in a nucleoid.
Prokaryotes can carry extra-chromosomal DNA elements called plasmids, which are
usually circular. Plasmids enable additional functions, such as antibiotic resistance. The
presence of plasmid DNA in bacteria not only made bacteria a distinct from animal or
plant but also put the bacteria in the unique position as an important genetic engineering
tool.
Q3. Explain Mendelian’s genetics.
Answer: For thousands of years, farmers and herders have been selectively breeding
their plants and animals to produce more useful hybrids. It was somewhat of a hit or miss
process since the actual mechanisms governing inheritance were unknown. Knowledge of
these genetic mechanisms finally came as a result of careful laboratory breeding
experiments carried out over the last century and a half. By the 1890's, the invention of
better microscopes allowed biologists to discover the basic facts of cell division and
sexual reproduction. The focus of genetics research, then shifted to understanding what
really happens in the transmission of hereditary traits from parents to children. A number
of hypotheses were suggested to explain heredity, but Gregor Mendel, a little known
Central European monk, was the only one who got it more or less right. His ideas
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published in 1866 but largely went unrecognized until 1900, which was long after his
death. While Mendel's research was with plants, the basic underlying principles of
heredity that he discovered also apply to people and other animals because the
mechanisms of heredity are essentially the same for all complex life forms. Through the
selective cross-breeding of common pea plants (Pisum sativum) over many generations,
Mendel discovered that certain traits show up in offspring without any blending of
parental characteristics. For instance, the pea flowers are either purple or white--
intermediate colors do not appear in the offspring of cross-pollinated pea plants. Mendel
observed seven traits that were easily recognized and apparently only occurred in one of
two forms:
Flower color is purple or white;
Flower position is terminal
Stem length is long or short
Seed shape is round or wrinkled
Seed color is yellow or green
Pod shape is inflated or constricted
Pod color is yellow or green
This observation that these traits do not show up in offspring plants with intermediate
forms were critically important because the leading theory in biology at the time was that
inherited traits blend from generation to generation. Most of the leading scientists in the
19th century accepted this "blending theory." Charles Darwin proposed another equally
wrong theory known as "pangenesis". This held that hereditary "particles" in our bodies
are affected by the things we do during our lifetime. These modified particles were
thought to migrate via blood to the reproductive cells and subsequently could be inherited
by the next generation. This was essentially a variation of Lamarck's incorrect idea of the
"inheritance of acquired characteristics." Mendel picked common garden pea plants for
the focus of his research because they can be grown easily in large numbers and their
reproduction can be manipulated. Pea plants have both male and female reproductive
organs. As a result, they can either self-pollinate themselves or cross-pollinate with
another plant. In his experiments, Mendel was able to select cross-pollinate purebred
plants with particular traits and observe the outcome over many generations. This was the
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basis for his conclusions about the nature of genetic inheritance. He came to three
important conclusions from these experimental results (i)- That the inheritance of each
trait is determined by "units" or "factors" that are passed on to descendants unchanged
(these units are now called genes), (ii)- That an individual inherits one such unit from
each parent for each trait, and (iii) That a trait may not show up in an individual but can
still be passed on to the next generation. It is important to realize that, in this experiment,
the starting parent plants were homozygous for pea seed color. That is to say, they each
had two identical forms (or alleles) of the gene for this trait--2 yellows or 2 greens. The
plants in the F1 generation were all heterozygous. In other words, they each had inherited
two different alleles--one from each parent plant. It becomes clearer when we look at the
actual genetic makeup, or genotype, of the pea plants instead of only the phenotype, or
observable physical characteristics. Note that each of the F1 generation plants inherited a
Y allele from one parent and a G allele from the other. When the F1 plants breed, each
has an equal chance of passing on either Y or G alleles to each offspring. With all of the
seven pea plant traits that Mendel examined, one form appeared dominant over the
other. Which is to say, it masked the presence of the other allele? For example, when the
genotype for pea seed color is YG (heterozygous), the phenotype is yellow. However, the
dominant yellow allele does not alter the recessive green one in any way. Both alleles can
be passed on to the next generation unchanged.
Q4. Explain supercoiling in a DNA molecule.
Answer: One of the interesting characteristics of DNA is its ability to form a coil like
structure and this process of making coiling is called as DNA supercoiling. With DNA in
its "relaxed" state, a strand usually circles the axis of the double helix once every 10.4
base pairs, but if the DNA is twisted the strands become more tightly. If the DNA is
twisted in the direction of the helix, this is positive supercoiling, and the bases are held
more tightly together. If they are twisted in the opposite direction, this is negative
supercoiling, and the bases come apart more easily. In nature, most DNA has slight
negative supercoiling that is introduced by enzymes called topoisomerases. These
enzymes are also needed to relieve the twisting stresses introduced into DNA strands
during processes such as transcription and DNA replication.
Q5. Describe the role of DNA polymerase in replication.
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Answer: DNA polymerases are a family of enzymes that carry out all forms of DNA
replication. A DNA polymerase can only extend an existing DNA strand paired with a
template strand; it cannot begin the synthesis of a new strand. To begin synthesis of a
new strand, a short fragment of DNA or RNA, called a primer must be created, and
paired with the DNA template. Once a primer pairs with DNA to be replicated, DNA
polymerase synthesizes a new strand of DNA by extending the 3’ end of an existing
nucleotide chain, adding new nucleotides matched to the template strand one at a time via
the creation of phosphodiester bonds. The energy for this process of DNA polymerization
comes from two of the three total phosphates attached to each unincorporated base. Free
bases with their attached phosphate groups are called nucleoside triphosphates. When a
nucleotide is being added to a growing DNA strand, two of the phosphates are removed
and the energy produced creates a phosphodiester bond that attaches the remaining
phosphate to the growing chain. The energetic of this process also help explain the
directionality of synthesis if DNA were synthesized in the 3’ to 5’ direction, the energy
for the process would come from the 5' end of the growing strand rather than from free
nucleotides. DNA polymerases are generally extremely accurate, making less than one
error for every 107 nucleotides added. Even so, some DNA polymerases also have
proofreading ability; they can remove nucleotides from the end of a strand in order to
correct mismatched bases. If the 5’ nucleotide needs to be removed during proofreading,
the triphosphate end is lost. Hence, the energy source that usually provides energy to add
a new nucleotide is also lost.
Q 6 What are topoisomerases and helicases?
Answer: Topoisomerases are enzymes with both nuclease and ligase activity. These
proteins change the amount of supercoiling in DNA and some of these enzyme work by
cutting the DNA helix and allowing one section to rotate, thereby reducing its level of
supercoiling; the enzyme then seals the DNA break. Other types of these enzymes are
capable of cutting one DNA helix and then passing a second strand of DNA through this
break, before rejoining the helix. Topoisomerases are required for many processes
involving DNA, such as DNA replication and transcription. Helicases are proteins that
are a type of molecular motor. They use the chemical energy in nucleoside triphosphates,
predominantly ATP, to break hydrogen bonds between bases and unwind the DNA
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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double helix into single strands. These enzymes are essential for most processes where
enzymes need to access the DNA bases.
Q7. How does DNA methylation occur?
Answer: DNA methylation has been proven by research to be manifested in a number of
biological processes such as regulation of imprinted genes, X chromosome inactivation,
and tumor suppressor gene silencing in cancerous cells. It also acts as a protection
mechanism adopted by the pathogen DNA mainly bacterial against the endonuclease
activity that destroys any foreign DNA. The expression of genes is influenced by how the
DNA is packaged in chromosomes, in a structure called chromatin. Base modifications
can be involved in packaging, with regions that have low or no gene expression usually
containing high levels of methylation of cytosine bases. For example, cytosine
methylation produces 5-methylcytosine, which is important for X-chromosome
inactivation. The average level of methylation varies between organisms - the worm
Caenorhabditis elegans lacks cytosine methylation, while vertebrates have higher levels,
with up to 1% of their DNA containing 5-methylcytosine. Despite the importance of 5-
methylcytosine, it can deaminate to leave a thymine base, methylated cytosine are
therefore particularly prone to mutations. Other base modifications include adenine
methylation in bacteria and the glycosylation of uracil to produce the "J-base" in
kinetoplastids.
Q 8 What is a PCR?
Answer: Polymerase chain reaction (PCR) is a technique to amplify a single or few
copies of a piece of DNA across several orders of magnitude, generating thousands of
millions of copies of a particular DNA sequence. PCR is developed in 1983 by Kary
Mullis, and now it is most common and indispensable technique used in medical and
biological research labs for a variety of applications. These include DNA cloning for
sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of
hereditary diseases; the identification of genetic fingerprints, and the detection and
diagnosis of infectious diseases. The method relies on thermal cycling, consisting of
cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic
replication of the DNA. Primers which are basically short DNA fragments containing
sequences complementary to the target region along with a DNA polymerase are key
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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components to enable selective and repeated amplification. As PCR progresses, the DNA
generated is itself used as a template for replication, setting in motion a chain reaction in
which the DNA template is exponentially amplified. PCR can be extensively modified to
perform a wide array of genetic manipulations. Almost all PCR applications employ a
heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated
from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a
new DNA strand from DNA building blocks, the nucleotides, by using single-stranded
DNA as a template and DNA oligonucleotides which is also called as DNA primers,
which are required for initiation of DNA synthesis. The vast majority of PCR methods
use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined
series of temperature steps. These thermal cycling steps are necessary first to physically
separate the two strands in a DNA double helix at a high temperature in a process called
DNA melting. At a lower temperature, each strand is then used as the template in DNA
synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity
of PCR results from the use of primers that are complementary to the DNA region
targeted for amplification under specific thermal cycling conditions. Most PCR methods
typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some
techniques allow for amplification of fragments up to 40 kb in size. The PCR is
commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–
0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes
to achieve the temperatures required at each step of the reaction (see below). Many
modern thermal cyclers make use of the Peltier effect which permits both heating and
cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-
walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal
equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of
the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of
the reaction mixture or a ball of wax inside the tube.
Q9 How does forensic DNA profiling done with a PCR tool?
Answer: Forensic scientists can use DNA in blood, semen, skin, saliva or hair found at a
crime scene to identify a matching DNA of an individual, such as a perpetrator. This
process is called genetic fingerprinting, or more accurately, DNA profiling. In DNA
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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profiling, the lengths of variable sections of repetitive DNA, such as short tandem repeats
and minisatellites, are compared between people. This method is usually an extremely
reliable technique for identifying a matching DNA. However, identification can be
complicated if the scene is contaminated with DNA from several people. DNA profiling
was developed in 1984 by British geneticist Sir Alec Jeffreys, and first used in forensic
science to convict Colin Pitchfork in the 1988 Enderby murders case. People convicted of
certain types of crimes may be required to provide a sample of DNA for a database. This
has helped investigators solve old cases where only a DNA sample was obtained from the
scene. DNA profiling can also be used to identify victims of mass casualty incidents. On
the other hand, many convicted people have been released from prison on the basis of
DNA techniques, which were not available when a crime had originally been committed.
Section B: Multiple Choice
Q1 100 trillion
Q2 Leeuwenhoek
Q3 No
Q4 Plasmids
Q5 Mitochondria
Q6 True
Q7 Both contain their own genome
Q8 Fungi
Q9 True
Q10 True
Q11 Genes
Q12 Sugar
Q13 Positive
Q14 Protein synthesis
Q15 Ribosome
Q16 False
Q17 Polymerase
Q18 DNA mutation
Q19 Kary Mullis
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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Q20 DNA Section C: Critical Thinking
Answers are not provided in this section as the students are encouraged to write their
own viewpoints.
CHAPTER 3 PROTEINS & PROTEOMICS
Section A: Descriptive Type
Q1: Explain the function of proteins as enzymes.
Answer: The best-known role of proteins in the cell is as enzymes, which catalyze
chemical reactions. Enzymes are usually highly specific and accelerate only one or a few
chemical reactions. Enzymes carry out most of the reactions involved in metabolism, as
well as manipulating DNA in processes such as DNA replication, DNA repair, and
transcription. Some enzymes act on other proteins to add or remove chemical groups in a
process known as post-translational modification. About 4,000 reactions are known to be
catalyzed by enzymes. The rate acceleration conferred by enzymatic catalysis is often
enormous as much as 1017-fold increase in rate over the uncatalyzed reaction in the case
of orotate decarboxylase. The molecules bound and acted upon by enzymes are called
substrates. Although enzymes can consist of hundreds of amino acids, it is usually only a
small fraction of the residues that come in contact with the substrate, and an even smaller
fraction 3 to 4 residues on average that are directly involved in catalysis. The region of
the enzyme that binds the substrate and contains the catalytic residues is known as the
active site.
Q2. Explain the role of proteins in cell signaling.
Answer: Many proteins are involved in the process of cell signaling and signal
transduction. Some proteins, such as insulin, are extracellular proteins that transmit a
signal from the cell in which they were synthesized to other cells in distant tissues. Others
are membrane proteins that act as receptors whose main function is to bind a signaling
molecule and induce a biochemical response in the cell. Many receptors have a binding
site exposed on the cell surface and an effectors domain within the cell, which may have
enzymatic activity or may undergo a conformational change detected by other proteins
within the cell. Antibodies are protein components of adaptive immune system whose
main function is to bind antigens or foreign substances in the body, and target them for
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
Courtesy of CRC Press/Taylor & Francis Group
Bacterial cell
Plasma membrane
CapsuleCell wall
Circular DNAPlasmid
Ribosome
Pili
Cytoplasm
Flagella
Human cell
CytoplasmMitochondriaNuclear pores
Rough endoplasmicreticulum Cell wall
Centrioles
Microfilaments
Plasma membrane
Golgi bodies
Microtubules
Smoothendoplasmic
reticulum
Nucleus
Nucleolus
Chromatin
Peroxisome
Chromatin
Lysosome
Ribosome
Figure 2.1 Prokaryotic cell and eukaryotic cell: Diagrammatic illustration.
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SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
Courtesy of CRC Press/Taylor & Francis Group
Protein channel
Hydrophobic region
Inside of cell(cytoplasm)
Outside of cell
Phospholipid bilayer
Hydrophilic region
Hydrophilic region
Figure 2.2 Cell membrane: Diagrammatic illustration.
002x002.eps
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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N
(a)
(b)
N
N N
O NH
H
H
H
H T
C
H
G
H
N
N
NA N
O
O
O
N
N
N
Adenine
GuanineCytosine
Thymine
N
N
N
Figure 2.3 (a) A GC base pair with three hydrogen bonds. (b) An AT base pair with two hydrogen bonds. Non-covalent hydrogen bonds between the pairs are shown as dashed lines.
002x003.eps
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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S
S
S
P
P
P
S
S
S
P
P
P
Hydrogen bonds
T
G
C
A
C
G
3΄
5΄
5΄
Phosphodiesterbonds
3΄
Purine
Pyrimidine
Figure 2.4 DNA base pairing.
002x004.eps
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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Double helix of DNA
S
S
S
A
G
T
P
P
P
S
S
S
T
C
A
P
P
PBase pairs
Hydrogen bondsNucleotide
Sugar phosphatebackbone
Figure 2.5 The structure of DNA: pieces of DNA are pairs of molecules, which entwine like vines to form a double helix. DNA strands are composed of four nucleo-tide subunits. These are adenine (A), thymine (T), cytosine (C), and guanine (G). Each base forms hydrogen bonds readily to only one other—A to T and C to G. The entire nucleotide sequence of each strand is complementary to that of the other.
002x005.eps
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
Courtesy of CRC Press/Taylor & Francis Group
Relaxed
Positive
SupercoilUncoilloop
Negative
Figure 2.6 DNA supercoiling.
002x006.eps
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
Courtesy of CRC Press/Taylor & Francis Group
S
S
S
U
A
C
P
P
P
S
S
S
A
U
G
P
P
PBase pairs
Hydrogen bonds
Sugar phosphatebackbone
Figure 2.7 RNA structure.
002x007.eps
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
Courtesy of CRC Press/Taylor & Francis Group
ACCUGCU
U U
U
CCGGU
TGG
AG
C
UA C
A
GGGCGUG
GGCCD
C C
GG
G G G
G
D A G CC
DC
GG
G
GGG
ICGI
CCC
CU
AA
UU
C3́
T CG loopD loop
Anticodon
5́
CAmino acid
Anticodon loop
Figure 2.8 Transfer RNA (tRNA) structure.
002x008.eps
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
Courtesy of CRC Press/Taylor & Francis Group
mRNA Small rRNA subunit
Psite
Asite
Large rRNA subunit
P A
Polypeptide chainNew amino acid added
5́ 3́
Codons
tRNAs
Figure 2.9 Ribosomal RNA (rRNA).
002x009.eps
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
Courtesy of CRC Press/Taylor & Francis Group
Parent chromosomes
Chromosomes replicate
Like chromosomes pair upand swap sections of DNA
Chromosomepairs divide
Daughter nucleidivide again
Figure 2.10 Stages of meiosis.
002x010.eps
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46 chromosomesInterphase
Doubling of chromosomesProphase
Nucleus dissolvedand microtubules attach to
centromeresPrometaphase
Chromosomes align atmiddle of cellMetaphase
Separation ofchromosomesAnaphase
Microtubules disappearand start to divide Telophase
Daughter cells with46 chromosomesCytokinesis
Figure 2.11 Stages of mitosis.
002x011.eps
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
Courtesy of CRC Press/Taylor & Francis Group
1.
Parental DNA
Single-strand binding protein stabilize the
unwound parental DNA
The leading strand is synthesizedcontinuously in the 5 3direction by DNA polymerase
3.
4.
5.
Replication fork
Primase
DNA primer
DNA ligase
DNA polymerase
Okazaki fragmentbeing made
3́
5́ 5́
3́
3́
5́
The lagging strand issynthesized discontinuously
After RNA primer is replaced by DNA,DNA ligase joins the Okazaki fragment to the growing strand
Helicases unwind the parental double helix
2.
Figure 2.12 DNA replication process.
002x012.eps
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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A T TC5́ 3́
G T A A3́ 5́
Eco RI
DNA fragments removed
C5́ 3́
G T A A3́ 5́
C
G T A A
DNA fragments join at sticky ends
RecombinantDNA
Figure 2.13 Role of restriction enzyme in making recombinant DNAATTCCC5.
002x013.eps
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
Courtesy of CRC Press/Taylor & Francis Group
O O
N
NH2 NH2
N
N
N
CH3
5΄-CpG-3΄3΄-GpC-5΄
DNAmethyl transferases
Cancer DNA
Normal DNA
Methylated CpG site
Unmethylated CpG site
Exon A Exon B
Exon A Exon B
Figure 2.14 DNA methylation.
002x014.eps
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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Mutation point
Mutated DNA sequence Normal DNA sequence
T
T
GG
A
A
C
GGC
C
TG
GG
CC
C
Figure 2.15 DNA mutation.
002x015.eps
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DNA sample
Forward and reverse primers
Sample preparation
PCR machine
Multi-�cation of DNAhappened in the PCR
Sample loading on gel
Gel electrophoresisGel documentation system
Final PCR product
Figure 2.16 Steps in the PCR.
002x016.eps
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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G
GG
G
C5 ΄
3΄
3΄
3΄
3΄
3΄
3΄ 3΄
3΄ 3΄
3΄
5΄
5΄
5΄
5΄ 5΄
5΄ 5΄
5΄
5΄
5΄
5΄
3΄3΄
C C
CG G
G
T
T T TA
A AA
Multiple copies of DNA
C CC GT T TACG GT A AA C C C GT T TA
CGT A AA
C C C
CG G
G
T
T T TA
A AA
Primers binds to template DNA strands at 55°C
Separation of DNA strands due to heat at 95°C
Multiplication of DNA strands using Taq DNApolymerase at 72°C
Multiple copies of DNA
G
Figure 2.17 Amplification of DNA fragment.
002x017.eps
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
Major fields of biotechnology
Title in here1
Chapter # 1
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
Major fields of biotechnology
Title in here2
• Cell is the building block of human body and it is critical to understand its structure and function so that better treatment modalities can be achieved.
• There is similarity and difference of intracellular and extracellular aspects of cells of prokaryotic and eukaryotic origin.
Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
CELL BIOLOGY
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
Major fields of biotechnology
Title in here3
• The cell is the structural and functional unit of all known living organisms.
• It is the smallest unit of a living organism, and is often called the building
block of life.
• Humans have approximately 100 trillion or 1014 cells, an example of a
multicellular organism. On the other hand, a single-celled bacterium is called
unicellular.
• A typical cell size is 10 micrometer while a typical cell mass is 1 nanogram.
All animals and plants are made of cells and such concept was originally
coined by Aristotle (384-322 BC).
Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
CELL AS A BUILDING BLOCK OF LIFE
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
Major fields of biotechnology
4
• All animals and plants are made of cells and such concept was originally
coined by Aristotle (384-322 BC).
• In 1665, Robert Hooke observed for the first time the structure of a cell under
a very primitive microscope.
• In 1674, Antonie van Leeuwenhoek discovered cells with structural
organization within the cell.
• In 1824, H.J. Dutrochet, a French scientist, gave the idea of the cell theory
Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
CELL AS A BUILDING BLOCK OF LIFE
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
Major fields of biotechnology
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• There are two different types of cells, the eukaryotic and prokaryotic cells.
• The prokaryote cell is simpler than a eukaryote cell, lacking a nucleus and
most of the other organelles of eukaryotes.
• There are two kinds of prokaryotes, bacteria and archaea, These prokaryotes
share a similar overall structure
• Eukaryotic cells are about 10 times the size of a typical prokaryote
• The major difference between prokaryotes and eukaryotes is that eukaryotic
cells contain membrane-bound compartments in which specific metabolic
activities take place
Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
CLASSIFICATION OF CELL
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
Title in here6Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
ORGANIZATION OF A CELL
External Organization
• Cell membrane• Cell capsule• Flagella
Internal Organization
Cytoplasmic Parts
• Mitochondria• Chloroplast• Ribosome• Endoplasmic reticulum• Golgi apparatus• Lysosomes and
Peroxisomes • Centrosomes• Vacuoles • Cytoskeleton
Nuclear Parts
• DNA• RNA• mRNA• tRNA• rRNA• snRNA• Nucleoli• Chromatins
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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Chapter # 2
MACRO-MOLECULES OF BODY
• Living cells are primarily made up of water, a number of other molecules are also abundant within a cell such as macromolecules.
• Macromolecules provide structural support, store fuel, store and retrieve genetic information, and speed up biochemical reactions.
• There are four major types of macromolecules that play important functions in the life a cell:
• PROTEINS, • CARBOHYDRATES, • NUCLEIC ACID, AND • LIPIDS
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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In most living organisms (except for viruses), genetic information is stored in DNA, which resides in the nucleus of living cells. It gets its name from the sugar
molecule contained in its backbone (deoxyribose). However, it gets its significance from its unique structure. Four different nucleotide bases occur in DNA: adenine
(A), cytosine (C), guanine (G), and thymine (T).
Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
DNA STRUCTURE
S
S
S
P
P
P
S
S
S
P
P
P
Hydrogen bonds
T
G
C
A
C
G
3’
5’
5’
3’
Phosphodiesterbonds
purine
pyrimidine
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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Ribonucleic acid (RNA) is a biologically important type of molecule that consists of a long chain of nucleotide units. RNA and DNA are both nucleic acids, but differ in three main ways. First, unlike DNA which is double-stranded, RNA is a single-stranded molecule in most of its biological roles and has a much shorter chain of nucleotides. Second, while DNA contains deoxyribose, RNA contains ribose, (there is no hydroxyl group attached to the pentose ring in the 2' position in DNA).
Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
RNA STRUCTURE
S
S
S
U
A
C
P
P
P
S
S
S
A
U
G
P
P
PBase pairs
Hydrogen bonds
Sugar phosphatebackbone
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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Transfer RNA (tRNA) is a small RNA molecule (usually about 74-95 nucleotides) that transfers a specific active amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation
Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
TRANSFER RNA STRUCTURE3’
5’
Amino acid
D loop
Anticodon loopAnticodon loop
Anticodon
T CG loop
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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Ribosomal RNA (rRNA) is the central component of the ribosome, the protein manufacturing machinery of all living cells. The function of the rRNA is to provide a mechanism for decoding mRNA into amino acids and to interact with the tRNAs during translation by providing peptidyl
transferase activity. The tRNA then brings the necessary amino acids corresponding to the appropriate mRNA codon
Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
RIBOSOMAL RNA STRUCTURE
mRNA
Small rRNA subunit
P site
Asite
large rRNA subunitP A
polypeptide chainNew amino acid added
5’ 3’
codons
tRNAs
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
Title in here12Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
CELL DIVISION
• All living cells undergo cell division in order to multiply and grow and there are two ways the cell can be divided such as meiosis and mitosis
• When cells are divided to yield two daughter cells, the genetic material must be divided equally so that each daughter cell contains identical DNA copies.
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Meiosis is a process of reduction division in which the number of chromosomes per cell is reduced to half. In animals, meiosis always results in the formation of gametes, while in other organisms it can give rise to spores.
Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
MEIOSIS
Parent chromosomes
Chromosomes Replicate
Like chromosomes pair up& swap sections of DNA
Chromosomes pairs divide
Daughter nuclei divide again
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Mitosis is the process in which a eukaryotic cell separates the chromosomesin its cell nucleus into two identical sets in two daughter nuclei. It is generally followed immediately by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two daughter cells containing roughly equal shares of these cellular components
Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
MITOSISInterphase
Prophase
Prometaphase
Metaphase
Anaphase
Telophase
Cytokinesis
46 chromosomes
Doubling of chromosomes
Nucleus dissolved and microtubules attach to centromeres
Chromosomes align at Middle of cell
Separation of chromosomes
Microtubules disappearand start to divide
Daughter cells with 46 chromosomes
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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In a cell, DNA replication begins at specific locations in the genome, called "origins". Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork
Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
DNA REPLICATION PROCESS
1 DNA Unwinding
Parental DNA
2 protein stabilize
the unwound parental DNA
3 DNA synthesis by
DNA polymerase
4 The lagging stand is synthesized
discontinuously
5 Okazaki fragment to
the growing strand
Replication fork
Primase
DNA primer
DNA ligase
DNA polymerase
Okazaki Fragment being made
5’
3’
3’
5’
5’
3’
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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A restriction enzyme is an enzyme that cuts DNA at specific recognition nucleotidesequences (with Type II restriction enzymes cutting double-stranded DNA) known as restriction sites
Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
ROLE OF RESTRICTION ENZYME
A T T C CC5 ‘ 3 ‘
G T A A G G
3 ‘ 5 ‘
CC5 ‘ 3 ‘
G T A A G G
3 ‘ 5 ‘
C
G
C5 ‘
G T A A G
3 ‘
Eco RI
DNA fragments removed
DNA fragments join at sticky ends
RecombinantDNA
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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DNA methylation is one such post-synthesis modification. DNA methylation has been proven by research to be manifested in a number of biological processes such as regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in cancerous cells
Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
DNA METHYLATION
O
N
NH2
NO
N
NH2
N
CH3
5’-CpG-3’3’-GpC-5’
DNAMethyl transferases
Cancer DNA
Normal DNA
Methylated CpG site
Unmethylated CpG site
Exon A Exon B
Exon A Exon B
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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DNA mutation is a change in the sequence of DNA. It can be caused by copying errors in the genetic material during cell division, by exposure to ultraviolet/ionizing radiation, chemical mutagens, or viruses, or by cellular processes such as hyper-mutation. It can also be induced by the organism itself.
Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
DNA MUTATION PROCESS
X-rayexposure
DNA Before
DNAAfter
StructuralChanges
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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Chapter # 2
DNA AMPLIFICATION BY POLYMERASE CHAIN REACTION (PCR)
Figure 2.18DNA Mutation
DNA sample
Reverse Primer
Forward Primer
PCR machine Sample loading on gel
Gel electrophoresisGel documentation system
Final PCR Product
Sample preparation
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
Title in here20Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
DNA MUTATION PROCESS
A T T C C G TC5 ‘ 3 ‘
G T A A G G C A3 ‘ 5 ‘
A T T C C G TC5 ‘ 3 ‘
G T A A G G C A3 ‘ 5 ‘
Separation of DNA strands due to heat at 95 degree C
Primers binds to Template DNA strands at 55 degree C
5 ‘ 3 ‘
3 ‘ 5 ‘
5’3’5’ 3’
A T T C C G TC5 ‘ 3 ‘G T A A G G C A
3 ‘ 5 ‘
A T T C C G TC5 ‘ 3 ‘G T A A G G C A
3 ‘ 5 ‘
Multiple copies of DNAMultiple copies of DNA
Multiplication of DNA strands using Taq DNA Polymerase at 72 degree C
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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Q1. What is the cell theory?
Q2. Describe the characteristics of a prokaryotic cell.
Q3. Explain Mendelian’s genetics.
Q4. Explain supercoiling in DNA molecule.
Q5. Describe the role of DNA polymerase in replication.
Q6. What are topoisomerases and helicases?
Q7. How does DNA methylation occur?
Q8. What is a polymerase chain reaction?
Q9. How does forensic DNA profiling done with PCR tool?
Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
FIND A SOLUTION FOR A PROBLEM
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN
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Q1 In order to identify the real culprit among a group of crime suspects, what
technique can be used to establish the identity of the culprit? Explain with
suitable examples.
Q2 Is it possible to study the genetic information of an individual by working
with mRNA only? Explain.
Q3 What would be the status of gene expression in case mRNA is not available?
Q4 What will happen if nuclear DNA is circular in shape and mitochondrial
DNA is linear in shape?
Biotechnology Fundamentals by Firdos Alam Khan
Chapter # 2
CRITICAL THINKING
SOLUTIONS MANUAL FOR BIOTECHNOLOGY FUNDAMENTALS 2ND EDITION KHAN