Download Ans8. Anaerobic Respiration/ Fermentation

Ques8. What do you understand by anaerobic respiration or fermentation?
Ans8. Anaerobic Respiration/ Fermentation - Oxidation of substances in absence of
atmospheric oxygen is known as anaerobic respiration. Here end products like ethanol or lactic acids are
produced and CO2 is released. The anaerobic pathways are important and are the sole source of ATP for
many anaerobic bacteria. Eukaryotic cells also resort to anaerobic pathways if their oxygen supply is
low. For example, when muscle cells are working very hard and exhaust their oxygen supply, they
utilize the anaerobic pathway to lactic acid to continue to provide ATP for cell function.
C6H12O6 → 2C2H5OH + 2CO2
+ 56 KCal ↑
Glycolysis itself yields two ATP molecules, so it is the first step of anaerobic respiration. Pyruvate, the
product of glycolysis, can be used in fermentation to produce ethanol and NAD+ or for the production of
lactate and NAD+. The production of NAD+ is crucial because glycolysis requires it and would cease
when its supply was exhausted, resulting in cell death.
Fig- Anaerobic Respiration/ Fermentation
Ques9. Explain TCA/ Krebs cycle in detail with figure.
Ans9. The Krebs / TCA Cycle - The tricarboxylic acid cycle (TCA cycle) is a series of enzymecatalyzed chemical reactions that form a key part of aerobic respiration in cells. This cycle is also called
the Krebs cycle (as studied by Hans Kreb, 1937).
Fig- TCA / Krebs Cycle
The cycle starts with pyruvate, which is the end product of glycolysis, the first step of all types of cell
respiration. The steps are The pyruvic acid produced during glycolysis enters into the mitochondrial matrix and gets
converted to 2 carbon acetylCoA.

AcetylCoA is added to 4 carbon oxaloacetic acid to form citric acid by enzyme citrate
synthetase.

Citric acid is dehydrated to form cis-aconitic acid in presence of enzyme aconitase.

Cis aconitic acid reacts with one molecule of water to form isocitric acid.

Isocitric acid is oxidized to form oxalosuccinic acid by isocytric dehydrogenase enzyme.

Oxalosuccinic acid is decarboxylated to α ketoglutaric acid by oxalosuccinic decarboxylase. CO 2
is released.

α ketoglutaric acid is oxidatively decarboxylated to form succinyl CoA by α ketoglutaric
dehydrogenase. CO2 is released and NAD+ is reduced to NADH + H+.

succinyl CoA is hydrolysed to succinic acid in presence of enzyme succinic thiokinase.

succinic acid is oxidized to form fumaric acid in the presence of enzyme succinic
dehydrogenase.

A molecule of H2O is added to fumaric acid to form malic acid by enzyme fumerase.

Malic acid is oxidized to oxaloacetic acid in presence of enzyme malic dehydrogenase.
Ques10. Describe structure and function of Deoxyribonucleic Acid (DNA).
Ans10. Structure of DNAA DNA molecule consists of two long polynucleotide chains composed of four types of nucleotide
subunits. Each of these chains is known as a DNA chain or a DNA strand. Hydrogen bonds between the
base portions of the nucleotides hold the two chains together. Nucleotides are composed of a five-carbon
sugars to which are attached one or more phosphate groups and a nitrogen-containing base. In the case
of the nucleotides in DNA, the sugar is deoxyribose attached to a single phosphate group (hence the
name deoxyribonucleic acid) and the base may be either adenine (A), cytosine (C), guanine (G), or
thymine (T). The nucleotides are covalently linked together in a chain through the sugars and
phosphates, which thus form a “backbone” of alternating sugar-phosphate-sugar-phosphate. Because
only the base differs in each of the four types of subunits, each polynucleotide chain in DNA is
analogous to a necklace strung with four types of beads (the four bases A, C, G, and T). These same
symbols (A, C, G, and T) are also commonly used to denote the four different nucleotides—that is, the
bases with their attached sugar and phosphate groups. The arrowheads at the ends of the DNA strands
indicate the polarities of the two strands, which run anti parallel to each other in the DNA molecule.
Fig- Structure of double helical anti parallel bipolar DNA
Function of DNAAt each cell division, the cell must copy its genome to pass it to both daughter cells. Because each strand
of DNA contains a sequence of nucleotides that is exactly complementary to the nucleotide sequence of
its partner strand, each strand can act as a template for the synthesis of a new complementary strand. In
other words, if we designate the two DNA strands as S and S′, strand S can serve as a template for
making a new strand S′, while strand S′ can serve as a template for making a new strand S. Thus, the
genetic information in DNA can be accurately copied by a simple process in which strand S separates
from strand S′ and each separated strand then serves as a template for the production of a new
complementary partner strand that is identical to its former partner.
Fig- Replication of DNA
The enzymes participating in the replication process areHelicase- cleaves or unwinds two DNA strands
DNA Polymerase- arranges complimentary monomeric units of DNA template.
Ligase- unites monomeric units to complete the newly formed strands
The ability of each strand of a DNA molecule to act as a template for producing a complementary strand
enables a cell to copy, or replicate, its genes before passing them on to its descendants.
Ques11. What is Cricks Central Dogma? Describe the process of protein synthesis in
detail.
Ans11. Crick's Central Dogma: Information flow (with the exception of reverse transcription) is
from DNA to RNA via the process of transcription, and thence to protein via translation. Transcription is
the making of an RNA molecule from a DNA template. Translation is the construction of an amino acid
sequence (polypeptide) from an RNA molecule.
Transcription- In transcription an mRNA chain is generated, with one strand of the DNA double
helix in the genome as a template. This strand is called the template strand. The DNA strand is read in
the 3' to 5' direction and the mRNA is transcribed in the 5' to 3' direction by the RNA polymerase.
Transcription occurs in the cell nucleus, where the helical DNA is held. This DNA is "unzipped" by the
enzyme helicase, leaving the single nucleotide chain open to be copied. RNA polymerase reads the
DNA strand from 3-prime (3') end to the 5-prime (5') end, while it synthesizes a single strand of
messenger RNA in the 5'-to-3' direction. The general RNA structure is very similar to the DNA
structure, but in RNA the nucleotide uracil takes the place that thymine occupies in DNA. The single
strand of mRNA leaves the nucleus through nuclear pores and migrates into the cytoplasm.
Translation- The synthesis of proteins is known as translation. Translation occurs in the cytoplasm,
where the ribosomes are located. Translation proceeds in four phases: activation, initiation, elongation,
and termination.
STEP 1: Activation: In activation, the correct amino acid (AA) is joined to the correct transfer RNA
(tRNA). The AA is joined by its carboxyl group to the 3' OH of the tRNA by an ester bond. When the
tRNA has an amino acid linked to it, it is termed "charged".
STEP 2: Initiation: Initiation Codon=AUG (Methionine) & Anticodon=UAC
In the cytoplasm, protein synthesis is actually initiated by the AUG codon on mRNA. The AUG codon
signals both the interaction of the ribosome with m-RNA and also the tRNA with the anticodons (UAC).
The tRNA which initiates the protein synthesis has N-formyl-methionine attached.
STEP 3: Elongation: Elongation of the peptide begins as various tRNA's read the next codon and a
correct match with the anticodons of a tRNA has been found.
Step 4: Elongation and Termination: Termination / Stop Codons=UAA, UAG, UGA
When the stop signal on mRNA is reached, the protein synthesis is terminated.
Fig- Protein Synthesis- Transcription and Translation
Ques12. Detail out various steps involved in mitotic cell division of eukaryotes.
Ans12. Mitosis or Asexual Reproduction- The cell division process that produces new cells for
growth, repair and the general replacement of older cells is called mitosis or asexual reproduction. In
this process, a somatic cell divides into two complete new cells that are identical to the original one.
Human somatic cells go through the 6 phases of mitosis in 1/2 to 1 & 1/2 hours, depending on the kind
of tissue being duplicated.
The process of cell division, called cell cycle, has four major parts called phases. The first part, called
G1 phase is marked by synthesis of various enzymes that are required for DNA replication. The second
part of the cell cycle is the S phase, where DNA replication produces two identical sets of
chromosomes. The third part is the G2 phase. Significant protein synthesis occurs during this phase,
mainly involving the production of microtubules, which are required during the process of division,
called mitosis.
The fourth phase, M phase, consists of nuclear division (karyokinesis) and cytoplasmic division
(cytokinesis), accompanied by the formation of a new cell membrane. The M phase has been broken
down into several distinct phases, sequentially known as prophase, prometaphase, metaphase, anaphase
and telophase leading to cytokinesis.
Mitosis only occupies a fraction of the cycle. The rest of the time-phases G1 through G2—is known as
interphase.
Phases of Mitosis-Cell Replication- Process is illustrated here with the help of only four diploid
chromosomes.
1. Interphase
DNA has replicated, but has not formed the condensed structure of chromosome. They remain as loosely
coiled chromatin.
The nuclear membrane is still intact to protect the DNA molecules from undergoing mutation.
Fig- Different phases of Mitosis
2. Prophase
The DNA molecules progressively shorten and condense by coiling, to form chromosomes. The
nuclear membrane and nucleolus are no longer visible.
The spindle apparatus migrate to opposite poles of the cell.
3. Metaphase
The spindle fibres attach themselves to the centromeres of the chromosomes and align the chromosomes
at the equatorial plate.
4. Anaphase
The spindle fibres shorten and the centromere splits, separated sister chromatids are pulled along behind
the centromeres.
5. Telophase
The chromosomes reach the poles of their respective spindles. Nuclear envelope reforms before the
chromosomes uncoil. The spindle fibres disintegrate.
6. Cytokinasis
This is the last stage of mitosis. It is the process of splitting the daughter cells apart. A furrow forms and
the cell is pinched in two. Each daughter cell contains the same number and same quality of
chromosomes.
Ques13. Give a brief description of PCR.
Ans13. POLYMERASE CHAIN REACTION (PCR) –
In 1985, a process was reported by which specific portions of the sample DNA can be amplified almost
indefinitely. This has revolutionized the whole field of DNA study. The process, the polymerase chain
reaction (PCR), mimics the biological process of DNA replication, but confines it to specific DNA
sequences of interest.
Fig- Polymerase Chain Reaction (PCR)
In this process, the DNA sample is denatured into the separate individual strands. Two DNA primers are
used to hybridize to two corresponding nearby sites on opposite DNA strands in such a fashion that the
normal enzymatic extension of the active terminal of each primer (that is, the 3’ end) leads toward the
other primer. In this fashion, two new copies of the sequence of interest are generated. Repeated
denaturation, hybridization, and extension in this fashion produce an exponentially growing number of
copies of the DNA of interest. The denaturation is generally performed by heating, and in this case
using, replication enzymes that are tolerant of high temperatures (Taq DNA polymerase). This process
can produce a million-fold or greater amplification of the desired region in 2 hours or less.
With the invention of the polymerase chain reaction (PCR) technique, DNA profiling took huge strides
forward in both discriminating power and the ability to recover information from very small (or
degraded) starting samples. PCR greatly amplifies the amounts of a specific region of DNA, using
oligonucleotide primers and a thermostable DNA polymerase. The PCR method is readily adaptable for
analyzing VNTRs. In recent years, research in human DNA quantitation has focused on new "real-time"
quantitative PCR (qPCR) techniques. Quantitative PCR methods enable automated, precise and high
through put measurements.
Ques14. Write an explanatory note on electrophoresis.
Ans14. ELECTROPHORESIS – Nucleic acid electrophoresis is an analytical technique used to
separate DNA or RNA fragments by size and reactivity. Nucleic acid molecules which are to be
analyzed are set upon a viscous medium, the gel, where an electric field induces the nucleic acids to
migrate toward the anode, due to the net negative charge of the sugar-phosphate backbone of the nucleic
acid chain. The separation of these fragments is accomplished by exploiting the mobility’s with which
different sized molecules are able to pass through the gel. Longer molecules migrate more slowly
because they experience more resistance within the gel. Because the size of the molecule affects its
mobility, smaller fragments end up nearer to the anode than longer ones in a given period. After some
time, the voltage is removed and the fragmentation gradient is analyzed. For larger separations between
similar sized fragments, either the voltage or run time can be increased. Extended runs across a low
voltage gel yield the most accurate resolution. Voltage is, however, not the sole factor in determining
electrophoresis of nucleic acids.
The nucleic acid to be separated can be prepared in several ways before separation by electrophoresis. In
the case of large DNA molecules, the DNA is frequently cut into smaller fragments using a DNA
restriction endonuclease (or restriction enzyme). In other instances, such as PCR amplified samples,
enzymes present in the sample that might affect the separation of the molecules are removed through
various means before analysis. Once the nucleic acid is properly prepared, the samples of the nucleic
acid solution are placed in the wells of the gel and a voltage is applied across the gel for a specified
amount of time. The DNA fragments of different lengths are visualized using a fluorescent dye specific
for DNA, such as ethidium bromide. The gel shows bands corresponding to different nucleic acid
molecules populations with different molecular weight.
The types of gel most commonly used for nucleic acid electrophoresis are agarose (for relatively long
DNA molecules) and polyacrylamide (for high resolution of short DNA molecules, for example in DNA
sequencing). Gels have conventionally been run in a "slab" format such as that shown in the figure, but
capillary electrophoresis has become important for applications such as high-throughput DNA
sequencing. Capillary electrophoresis results are typically displayed in a trace view called an
electropherogram.
Factors affecting migration of nucleic acids
1. The most important factor is the length of the nucleic acid molecule, smaller molecules travel
faster.
2. But conformation of the nucleic acid molecule, such as % single strand, supercoiling, etc., is also
a factor. When analyzing molecules by size, it is most convenient to analyze only linear
molecules to avoid this problem, e.g. DNA fragments from a restriction digest, linear DNA PCR
products, or RNAs.
3. Increasing the agarose or polyacrylamide concentration of a gel reduces the migration speed and
enables separation of smaller nucleic acid molecules.
4. The higher the voltage, the faster the DNA moves. But voltage is limited by the fact that it heats
the gel and ultimately causes it to melt. High voltages also decrease the resolution (above about 5
to 8 V/cm).
Q15. What is Electron transport chain equation? Discuss in detail?
Ans15. Electron Transport Chain Equation:
To study electron transport, let us start with glucose oxidation. The equation for this process is:
Glucose + 6O2 = 6CO2 + 6H2O + 2823 kJ/mol
To closely see electron transport, this equation can be broken down further:
Glucose + 6O2 = 6CO2 + 24H+ + 24eand,
6O2 + 24H+ + 24e- = 12H2O
Electron Transport Chain Equation Explained:
• The electron transfer process connecting the above two equations is a multi-step process that harnesses
the liberated free energy to form ATP.
• The 24 electrons produced are absorbed by coenzymes NAD+ and FAD to form NADH and FADH2;
these electrons are then transferred to O2.
• These electrons then pass to the electron transport chain and participate in redox reactions before
reducing O2 to H2O.
• Thus, protons are expelled from the mitochondrion; the free energy stored in the change in pH drives
the synthesis of ATP.
• The free energy necessary to generate ATP is extracted from the energy released when NADH and
FADH2 release electrons (oxidation) by the electron transport chain.
• Electrons are carried from Complexes I and II to Complex III by coenzyme Q (CoQ), and from
Complex III to Complex IV by the peripheral membrane protein cytochrome.
• As electrons are shuttled between the four complexes, they lost energy is harnessed to generate ATP.
Location of ETS- Inside the Mitochondrion
• All the above steps take place in the mitochondrion.
• We focus on the transport of NADH across the inner mitochondrial membrane.
• The inner mitochondrial membrane lacks a NADH transport protein.
• Only electrons from cytosolic NADH are transported into the mitochondrion by a cytoplasmic
"shuttle" system.
• This shuttle system is called malate-aspartate shuttle, which functions in the heart, liver, kidney;
mitochondrial NAD+ is reduced by cytosolic NADH.
Mitochondrial NAD+ Reduction by Cytosolic NADH:
Phase A:
• Oxaloacetate reduced to NAD+ and malate; malate transported to mitochondrial matrix; malate then
oxidized to yield NADH and oxaloacetate.
Phase B:
• Oxaloacetate converted to aspartate; aspartate transported from matrix to cytosol; aspartate then
converted to oxaloacetate in cytosol.
Q16. With an application explain the process of DNA finger printing.
Ans16. A technique used by scientists to distinguish between individuals of the same species using only
samples of their DNA is called DNA finger printing. The process of DNA fingerprinting was invented
by Alec Jeffreys at the University of Leicester in 1985.
Stages of DNA Profiling:
Stage 1:
 Cells are broken down to release DNA If only a small amount of DNA is available it can be
amplified using the polymerase chain reaction (PCR)
Stage 2:
 DNA molecules are very long .They may consist of millions of base pairs
 In order to study the structure of DNA, the molecules are broken up into smaller fragments by
enzymes called restriction enzymes .
 Restriction enzymes do not break up the DNA molecule randomly but ‘cut’ it at particular sites.
Restriction fragments:
For example, a restriction enzyme called EcoR1* ‘recognises’ the base sequence CAATTC and
cuts it between the two As
Recognised
↑
Cut
C-C-G-C-A-G-C-T-G-T-C-A
A-T-T-C- T-C-T-C-C-G-G-A-T-C-C-AOther restriction enzymes cut the DNA in different places and so produce fragments which are easier to
analyse.
--C-C-G-C-A-G-C-T-G-T-C-A
↑
--C-C-G-C-A-G
C-T-G-T-C-A
A-T-T-C-T-C-T-C-C-G-G-A-T-C-C-C-A↑
A-T-T-C-T-C-C-G
G-A-T-C-C-C-A-
 The sections of DNA that are cut out are called restriction fragments.
 This yields thousands of restriction fragments of all different sizes because the base sequences
being cut may be far apart (long fragment) or close together (short fragment).
Gel electrophoresis:
The different sized fragments are separated by a process called gel electrophoresis. The
separation takes place in a sheet of a firm but jelly-like substance (a ‘gel’). Samples of the DNA
extracts are placed in shallow cavities (‘wells’) cut into one end of the gel. A voltage is applied
to opposite ends of the gel DNA has a negative charge and moves slowly towards the positive
end. The shorter fragments travel through the gel faster than the longer fragments.
Stage 3:
 A radioactive material is added which combines with the DNA fragments to produce a
fluorescent image.
 A photographic copy of the DNA bands is obtained.
Stage 4:
 The pattern of fragment distribution is then analysed.
Genetic fingerprint:
The pattern of bands in a gel electrophoresis is known as a genetic fingerprint or a ‘genetic profile’. If a
genetic fingerprint found in a sample of blood or other tissue at the scene of a crime matches the genetic
fingerprint of a suspect, this can be used as evidence. A DNA sample can be obtained from the suspect
using blood, cheek epithelial cells taken from the mouth lining or even the cells clinging to the root of a
hair.
DNA Profiling can solve crimes:
 The pattern of the DNA profile is then compared with those of the victim and the suspect.
 If the profile matches the suspect it provides strong evidence that the suspect was present at the
crime scene.

If the profile doesn’t match the suspect then that suspect may be eliminated from the enquiry.
Q17. Describe the male reproductive system in detail?
Ans17. Male reproductive system consists of the following:
A.
B.
C.
D.
Testis
Spermatogenesis
Male Reproductive Tract
Semen
A. Testis: The testis contain two compartments:
1. Seminiferous tubules (90% of weight): Sertoli cells, spermatogenesis, stimulated by FSH
2. Interstitial compartment: Leydig cells, stimulated by LH
B. Spermatogenesis: From spermatogonia (original stem cell in gonad) to spermatozoa. Cells
migrate from the embryonic yolk sac to the testes. In the seminiferous tubules they become
spermatogonia and then through a process called spermatogenesis the spermatogonia become
spermatids and then mature spermatozoa. The process from spermatids to spermatozoa is called
spermiogenesis.



Testosterone required for spermiogenesis in adult.
Later stages of spermatogenesis require FSH. Testosterone and FSH act on Sertoli cells which
probably release paracrine substances that stimulate spermatogenesis
Spermatozoa are non-motile in testes. They become motile and undergo other changes outside of
the testes.
Spermatozoa have 3 parts:
a. oval-shaped head: contains the DNA
b. midpiece or body: contains mitochondria for energy
c. tail: for swimming
C. Male Reproductive Tract- Testis→ epididymis→Vas
deferens→ejaculatory duct→urethra





Testes: formation of sperm
Epididymis: sperm maturation and sperm storage
Vas deferens: duct to transport spermatozoa towards the
seminal vesicles
Seminal vesicles: add secretions to form semen
Prostate gland: add secretions to semen
D. Semen
1. seminal vesicles: 60% of semen volume comes from seminal vesicles — contains fructose
2. Prostate gland: citric acid, calcium, coagulation proteins.
Q18. Describe the female reproductive system in detail?
Ans18. Female reproductive system consists of the following:
A.
B.
C.
D.
E.
Anatomy
Oocyte and Follicle Development
Menstrual Cycle
Contraceptive Methods
Fertilization
A. Anatomy




Ovaries: contain follicles which contain ova.
Accessory Sex Organs
Uterine or Fallopian tubes: ducts directly connected to uterus
Uterus: 3 layers perimetrium (connective tissue), myometrium (smooth muscle), endometrium
(epithelium)
B. Oocyte and Follicle Development
Period in Life
Number of Oocytes
5-6 months
6-7 million (oogonia)
Birth
1-2 million (primary oocytes)
Puberty
400,000
At 5 months of gestation there is a peak of about 6-7 million oogonia. After 5-6 months production of
oogonia stops and never resumes. These oogonia become primary oocytes by the end of gestation. At
puberty there are only about 400,000 primary oocytes. Only about 400 of these oocytes will actually
ovulate during a woman's lifetime.


Oocyte Development: oogonia (46 chromosomes) → primary oocyte (46 chromosomes) →
secondary oocyte (23 chromosomes) → zygote (if fertilized)
Follicular Development: primary follicles → secondary follicles → graafian follicle → corpus
luteum (after ovulation)
Types of Follicles:



Primary follicle: immature: primary oocyte + a single layer of follicular cells mature: primary
oocyte + a number of layers of follicular cells.
Secondary follicle: primary oocyte or secondary oocyte plus numerous layers of granulosa cells
and fluid filled vesicular cavities
Graafian follicle: secondary oocyte, arrested before second meiotic division, plus layers of
granulosa cells and a single large fluid filled cavity (antrum).
C. Menstrual Cycle
Changes in Ovary


Menstruation (Day 1 to Day 4or 5): steroid hormones lowest, ovaries contain primary follicles
Follicular Phase (Day 1 to Day 13, highly variable): FSH → growth of follicles, one becomes
mature graafian follicle, granulosa cells secrete estradiol
Positive feedback loop: (LH surge): estradiol→ GnRH→ LH secretion→ estradiol by ovaries


Ovulation: FSH, followed by LH surge causes rupture of graafian follicle, expulsion of
secondary oocyte into uterine tubes. Occurs about 24 hours after beginning of LH surge
Luteal Phase: empty follicle becomes corpus luteum, secretes estradiol and progesterone.
Changes in the endometrium



Proliferative Phase: proliferation of endometrium and increase in blood vessels (spiral arteries)
Secretory Phase: development of endometrium — thick, vascular, spongy — in preparation for
the embryo.
Menstrual
endometrium. Bleeding phase.
D. Contraceptive Methods
Conception is most likely to occur when intercourse takes place 1-2 days prior to ovulation.


Contraceptive pill: contains estrogen and progesterone, maintains negative feedback throughout
cycle, so no LH surge and therefore no ovulation.
Rhythm method: measure slight variations in temperature that normally occur just prior to
ovulation. Often hard to detect temperature changes.
E. Fertilization
Fertilization occurs in the uterine tubes.
1. Sperm capacitation, a series of changes which makes sperm fertile, occurs in the female tract.
Sperm can last up to 3 days in female reproductive tract.
2. Sperm fuses with ovulated oocyte in the uterine tube
3. Fusion of one sperm prevents other sperm from fertilizing oocyte
4. Zygote (diploid, 46 chromosomes) forms 12 hours after fertilization
5. The zygote begins dividing (cleavage)
6. Unfertilized oocyte will degenerate 12-24 hours after ovulation.
Fertilization cannot take place more than 1 day after ovulation. Since sperm live for 3 days in the female
reproductive tract, fertilization can occur up to 3 days prior to ovulation.
Blastocyst Formation



3 days after ovulation the embryo (8 cells at this point) enters the uterus
2 days later blastocyst forms
7 days after fertilization embryo is implanted in the uterine wall (endometrium)
Q19. Define gene cloning and the process of gene cloning?
Ans19. Gene cloning is the technique of recombinant DNA technology in which a desired gene of
interest having a striking characteristic feature is cloned. The gene may be selected because it appears to
influence the organism in a striking manner, or to determine the role of the gene in the organism. Genes
can be clones for industrial purposes, for instance the production of vaccines and insulin, or for research
purposes, to determine what the role of the gene is. Gene cloning requires a basic knowledge of the
gene's sequence, or flanking sequences. Genes can be cloned using polymerase chain reaction (PCR), if
the sequence is known, or by cutting genomic DNA with restriction enzymes (to create smaller chunks
of DNA). Usually, once a fragment containing gene has been identified using restriction enzymes, it is
sequenced and PCR is used to isolate the specific sequence within the fragment.
Procedure of gene cloning:
1) Firstly we must get the DNA we want in sufficient quantities to clone.
2) Next we decide on a vector. A vector is usually a piece of DNA that replicates in a bacterium.
Plasmids are usually used, they are small pieces of circular DNA that are often found in bacteria which
are self-replicating and are maintained in the cell in a stable and characteristic number of copies. Some
bacteria have very high numbers of plasmids which make them ideal cloning vectors.
Routinely in the lab a weakened strain of the bacteria E.coli is used for these experiments. If it is not
grown in the lab under special conditions it will quickly die.
3) Application of restriction digestion to cut the DNA.
Next we use restriction enzymes to cut out the gene we want from the amplified product. We use the
same enzymes to cut the vector so that the ends will be compatible. This means that if we use a "sticky
ends" restriction enzyme, then the DNA sequences of the trailing bits will be compatible and will want
to stick to each other. This makes it much easier to do. If we can't find a suitable site for a sticky end
restriction enzyme then we can use a "blunt-ended" one, which is a bit harder to make work.
Once the restriction is done the piece of DNA that you want to clone needs to be separated from the
parts you do not want this is done using gel electrophoresis as this will make it easier to clone (see
screening, below). This is usually done by running the digest out on a gel and physically cutting out the
fragment that you want with a scalpel.
4) Ligation Once both the vector and the target DNA have been cut we mix them together and add the
ligase enzyme. This enzyme ligates (connects) the phosphodiester backbone acting as a glue to stick the
ends together.
5) Transform bacteria: This just means add the DNA back into a bacterium using different popular ways.
6) Screening for 'positives'; those bacteria that have the cloned DNA in them.
Q20. Write short note on stem cells?
Ans20. Stem cells are undifferentiated biological cells that can differentiate into specialized cells and
can divide (through mitosis) to produce more stem cells. They are found in multicellular organisms. In
mammals, there are two broad types of stem cells: embryonic stem cells, which are isolated from the
inner cell mass of blastocysts, and adult stem cells, which are found in various tissues. In adult
organisms, stem cells and progenitor cells act as a repair system for the body, replenishing adult tissues.
In a developing embryo, stem cells can differentiate into all the specialized cells—ectoderm, endoderm
and mesoderm—but also maintain the normal turnover of regenerative organs, such as blood, skin, or
intestinal tissues.
There are three accessible sources of autologous adult stem cells in humans:
1. Bone marrow, which requires extraction by harvesting, that is, drilling into bone (typically the
femur or iliac crest),
2. Adipose tissue (lipid cells), which requires extraction by liposuction, and
3. Blood, which requires extraction through apheresis, wherein blood is drawn from the donor
(similar to a blood donation), passed through a machine that extracts the stem cells and returns
other portions of the blood to the donor.
Stem cells can also be taken from umbilical cord blood just after birth. Of all stem cell types, autologous
harvesting involves the least risk. By definition, autologous cells are obtained from one's own body, just
as one may bank his or her own blood for elective surgical procedures.