Medico Express 2nd year

MBBS
Block 5

ENDOCRINOLOGY
MODULE

BIOCHEMISTRY
GENETICS
EnR- Define the following terms: chromosome, allele (dominant Biochemistry Nomenclature
B- and recessive), gene, locus, heterozygote, homozygote, of genetics
009 hemizygous, autosome, genotype, phenotype, haploid and
diploid number of chromosomes, aneuploidy, proband,
proposita, pedigree, propositus, penetrance, codominance
and polygenic

GENETICS
DEFINITIONS OF INTRODUCTORY TERMS:
Term Definition
Genetics: The branch of biology that deals with heredity, especially the mechanisms of
hereditary transmission and the variation of inherited characteristics among similar
or related organisms
Chromosome • Genes are packaged in bundles called chromosomes.
• Humans have 23 pairs of chromosomes (for a total of 46).
• Of those, 1 pair is the sex chromosomes (determines whether you are male
or female)
• The other 22 pairs are autosomal chromosomes (determine the rest of the
body’s makeup).

Autosome: Chromosomes other than sex chromosomes are called autosomes. Humans have 22
pairs of autosomes
Gene: A gene is a distinct portion of a cell’s DNA.
Genes are coded instructions for making everything the body needs, especially
proteins.
Gene holds the information to build and maintain their cells and pass genetic traits
to offspring

Locus: Locus is defined as the location of a gene on a chromosome

Allele: Alleles are pairs or series of genes on a chromosome that determine the hereditary
characteristics
An example of an allele is the gene that determines hair color.

Dominant A dominant allele produces a dominant phenotype in individuals who have one copy
Allele of the allele, which can come from just one parent.
For example, If B is dominant, Bb will show the phenotype of B
Recessive For a recessive allele to produce a recessive phenotype, the individual must have
Allele two copies, one from each parent.
An individual with one dominant and one recessive allele for a gene will have the
dominant phenotype
Homozygous An organism in which 2 copies of genes are identical i.e. have the same alleles
Homozygous means you carry two genes that are the same for each trait. Example:
BB for brown eyes.
Heterozygous An organism that has different alleles of the gene or Heterozygous means you carry
two different genes for each trait
Example: Bb would also be brown eyes, though the small “b” would be the gene for
blue eyes. It doesn’t show up in the phenotype (outward appearance) because the
gene for brown eyes is dominant. If someone had blue eyes they would be
homozygous “bb”.
Hemizygous It occurs when only one allele is Present for a gene.
For Example, in Males, XY is present
Phenotype: A phenotype (from Greek painting, meaning "to show", and typos, meaning "type")
is the composite of an organism's observable characteristics or traits, such as its
morphology, development, biochemical or physiological properties, phenology,
behavior, and products of behavior (such as a bird’s nest)
Genotype Genotype refers to the Genetic makeup of a gene.
BB and Bb will have the same phenotype but different genotypes
Haploid Having 1n set chromosome is called haploid.
Diploid Having a 2n set of chromosomes is called diploid
Aneuploidy Aneuploidy is the presence of an abnormal number of chromosomes in a cell, for
example, a human cell having 45 or 47 chromosomes instead of the usual 46
Proband A person serving as the starting point for the genetic study of a family
Proposita The first identified female case of an inherited disease in a family
Pedigree A pedigree, as related to genetics, is a chart that diagrams the inheritance of a trait
or health condition through generations of a family
Propositus the family member chosen as the starting point in genealogical research, in the
investigation of an inheritable disease
Penetrance The extent to which a particular gene or set of genes is expressed in the phenotypes
of individuals carrying it is measured by the proportion of carriers showing the
characteristic phenotype.
Co- Different alleles of a gene that are both expressed in a heterozygous condition are
Dominance: called co dominance
Example: ABO Blood group
Polygenic A polygenic trait is a characteristic, such as height or skin color, that is influenced by
trait two or more genes
EnR-B- Discuss the structures of genes, how they are organized and Biochemistry Genes
010 regulated

GENE:
• Genes are functional units of heredity as they are made of DNA.
• The chromosome is made of DNA containing many genes. Every gene comprises a particular set
of instructions for a particular function or protein coding. Speaking in usual terms, genes are
responsible for heredity.
• The human cell contains 23 pairs of chromosomes.
• The trait is one of the characteristics determined by one or more genes.
• Abnormal genes and genes that are formed due to new mutations also result in certain traits.
• Genes vary in size depending on the code or the protein they produce.
• All cells in the human body contain the same DNA.
• The difference between the cells occurs due to the different types of genes that are turned on
and therefore produce a variety of proteins.
• Regulation of genes occurs differently, depending on the type of organisms- prokaryotic or
eukaryotic.
 EnR-B- Describe Mendelian Law of Segregation and Law of Biochemistry Mendelian
011 Independent Assortment. Law

MENDELIAN LAW OF SEGREGATION:

“According to the law of Segregation, the two co-existing alleles for each trait of an individual segregate
(separate) from each other at meiosis, so that each gamete receives only one of the two alleles. Allele
unites again at random fertilization of gamete when the zygote is formed”

LAW OF INDEPENDENT ASSORTMENT

“When two contrasting pairs of trait are followed in the same cross, their alleles assort
independently into gametes”
 EnR- Describe the patterns of inheritance characteristic of Biochemistry Patterns of
B-012 autosomal dominant, autosomal recessive, X- X-linked inheritance
dominant, X-linked recessive, and mitochondrial traits

Pattern of inheritance
AUTOSOMAL DOMINANT DISORDER:
• In autosomal dominant, only one copy of the allele present on the autosomal chromosome
causes the disease
• Affected males and females have an equal probability of passing on the trait to offspring.
• Affected individuals have one normal copy of the gene and one mutant copy of the gene, thus
each offspring has a 50% chance of inheriting the mutant allele.
• As shown in this pedigree, approximately half of the children of affected parents inherit the
condition and half do not.
• Individuals with these diseases usually have one affected parent

Inheritance Pattern:

• Typical mating pattern is a heterozygous affected individual with a homozygous unaffected

individual.
• Both sexes are affected equally.

Examples: Huntington’s Disease, Polycystic kidney disease, Marfan Syndrome

AUTOSOMAL RECESSIVE DISORDER:
• Recessive conditions are clinically manifest only when an individual has two copies of the
mutant allele.
• When just one copy of the mutant allele is present, an individual is a carrier of the mutation, but
does not develop the condition.
• Females and males are affected equally by traits transmitted by autosomal recessive
inheritance.
• When two carriers mate, each child has a 25% chance of being homozygous wild-type
(unaffected); a 25% chance of being homozygous mutant (affected); or a 50% chance of being
heterozygous (unaffected carrier).

Inheritance pattern

• Typical mating pattern is two heterozygous unaffected (carrier) individuals.

• The trait does not usually affect the parent, but siblings may show the disease Siblings have one
chance in four of being affected
• Both sexes affected equally.

Example: Xeroderma Pigmentosum, Sickle cell anemia, Cystic fibrosis

X-LINKED DOMINANT DISORDER:

• In X linked dominant disorder, the allele for the disease is located on X chromosome
• X-linked dominant disorders are clinically manifest when only one copy of the mutant allele is
present.
• Because the gene is located on the X chromosome, there is no transmission from father to son,
but there can be transmission from father to daughter (all daughters of an affected male will be
affected since the father has only one X chromosome to transmit).
• Children of an affected woman have a 50% chance of inheriting the X chromosome with the
mutant allele.
The pattern of inheritance:

• Typical mating pattern is two heterozygous unaffected (carrier) individuals.

• The trait does not usually affect the parent, but siblings may show the disease Siblings
have one chance in four of being affected
• Both sexes are affected equally.
Example: Rickets, Retinitis pigmentosa

X-LINKED RECESSIVE DISORDER :

• In X-linked recessive disorder, the allele is Present on the X chromosome

• It is more common in males because of carrying the affected X-chromosome from the
Carrier mother
• Women will only express disease when they have both effected X chromosome
• Disease usually passed on from the carrier mother
• An unaffected woman carries one copy of a gene mutation for an X-linked recessive
disorder. She has an affected son, an unaffected daughter who carries one copy of the
mutation, and two unaffected children who do not have the mutation.
Example: Duchenne Muscular dystrophy
EnR-B-013 Interpret genetic symbols as they appear in pedigrees Biochemistry Pedigrees
EnR- Analyze pedigree to determine the mode of inheritance of the Biochemistry Mode of
B- following traits: 1) X-linked recessive (Duchenne Muscular inheritance
014 dystrophy) 2) X-linked dominant (Rickets) 3) Autosomal
recessive (Xeroderma Pigmentosum) 4) Autosomal dominant
(Huntington’s Disease)) Mitochondrial disorder (Mitochondrial
diabetes)

PEDIGREE OF X-LINKED RECESSIVE DISORDER:

• In pedigrees that display X-linked recessive inheritance, sons of a carrier mother and an
unaffected father have a 50% chance of being affected, while daughters will not be affected, but
have a 50% chance of being carriers (heterozygotes).
• Sons of an affected mother will all be affected.
• There is no father-to-son transmission of X-linked traits.

PEDIGREE OF X-LINKED DOMINANT DISORDER:

• For this mode of inheritance, affected fathers will pass on the allele and condition to all of their
daughters.
• Sons of an affected, heterozygous mother have a 50% chance of being affected.
• For a daughter to be homozygous for the mutant allele, both of her parents must be affected by
the mutant trait.
• Thus all affected females on this chart are heterozygous in genotype.
AUTOSOMAL OF X-LINKED RECESSIVE DISORDER:
• If an affected individual in a pedigree has two unaffected parents, the condition is most likely
recessive.
• Additionally, if daughters in the pedigree have two unaffected parents, the condition is most
likely autosomal recessive.
• With autosomal recessive inheritance, males and females are equally likely to be affected.

AUTOSOMAL OF X-LINKED DOMINANT DISORDER:

• One copy of the mutant allele is sufficient to develop an autosomal dominant condition.
• In contrast, an individual must have two copies of the mutant allele to display an autosomal
recessive condition.
 EnR-B- Discuss different structural and numerical Biochemistry Chromosomal
015 chromosomal abnormalities. abnormalities

CHROMOSOMAL ABNORMALITIES

DOWN SYNDROME:
• Most common chromosomal disorder.
• Down syndrome is a chromosomal abnormality characterized by the presence of an extra copy
of genetic material on the 21st chromosome
• Trisomy 21 is caused by a meiotic nondisjunction (attach) event.
• With nondisjunction, a gamete (i.e., a sperm or egg cell) is produced with an extra copy of
chromosome 21; the gamete thus has 24 chromosomes
• When combined with a normal gamete from the other parent, the embryo now has 47
chromosomes, with three copies of chromosome 21.
• About 4% of cases are due to Robertsonian translocations.
• Maternal age has a strong influence

Other Trisomy Syndromes:

Trisomy 18: Edwards Syndrome.

Trisomy 13: Patau Syndrome.

CHROMOSOMAL INCIDENCE USUAL CLINICAL MANIFESTATIONS
ABERRATION AND
SYNDROME
Trisomy 21 (Down 1 in 800 Mental deficiency; brachycephaly, flat nasal bridge; upward slant to palpebral
syndrome)* fissures; protruding tongue; transverse palmar flexion crease; clinodactyly of
the fifth digit; congenital heart defects; gastrointestinal tract abnormalities
Trisomy 18 syndrome 1 in 8000 Mental deficiency; growth retardation; prominent occiput; short sternum;
(Edwards syndrome)† ventricular septal defect; micrognathia; low-set, malformed ears, flexed digits,
hypoplastic nails; rocker-bottom feet
Trisomy 13 syndrome 1 in 12,000 Mental deficiency; severe central nervous system malformations; sloping
(Patau syndrome)† forehead; malformed ears, scalp defects; microphthalmia; bilateral cleft lip
and/or palate; polydactyly; posterior prominence of the heels
*The importance of this disorder in the overall problem of mental deficiency is indicated by the fact that persons with Down
syndrome represent 10% to 15% of institutionalized individuals with severe mental retardation. The incidence of trisomy 21
at fertilization is greater than at birth; however, 75% of embryos are spontaneously aborted, and at least 20% are stillborn.
†Infants with this syndrome rarely survive beyond 6 months."
KLINEFELTER’S SYNDROME:
• Defined as Male Hypogonadism (does not produce enough testosterone), develops when there
are at least two X chromosomes and one or more Y chromosomes.
• Usual karyotype is 47, XXY. The extra X may be maternal or paternal.
• Results from nondisjunction of sex chromosome during meiosis
• Risk factors include advanced maternal age and a history of exposure to radiation in either
parent.

TURNER SYNDROME
• Primary hypogonadism in females
• Results from partial or complete monosomy of the X chromosome.
• The most common cause is the absence of one X chromosome.

CHROMOSOME SEX INCIDENCE+ USUAL CHARACTERISTICS

COMPLEMENT*
47,XXX Female 1 in 1000 Normal in appearance; usually fertile; 15% to 25% are
mildly mentally deficient
47,XXY Male 1 in 1000 Klinefelter syndrome: small testes, hyalinization of
seminiferous tubules; aspermatogenesis; often tall with
disproportionately long lower limbs. The intelligence of
siblings is less than normal. Approximately 40% of these
males have gynecomastia (see Fig. 20-9).
47, XYY Male 1 in 1000 Normal in appearance, usually tall, and often exhibit
aggressive behavior
*The numbers designate the total number of chromosomes, including the sex chromosomes shown after the
comma.
+Data from Hook EB, Hamerton JL: The frequency of chromosome abnormalities detected in consecutive newborn
studies; differences between studies; results by sex and by severity of phenotypic involvement. In Hook EB, Porter
IH, editors: Population cytogenetics: studies in humans, New York, 1977, Academic Press. More information is
provided by Nussbaum RL, McInnes RR, Willard HF: Thompson and Thompson genetics in medicine, ed 7,
Philadelphia, 2007, Saunders.
 EnR-B- Interpret the normal human karyotype in terms of the number Biochemistry Karyotypes
016 and structure of chromosomes.

Karyotype:
• The Particle array of chromosomes is called the Karyotype
• Normally, the human karyotype consists of 23 pairs of chromosomes: 22 pairs are called
autosomes, and the last pair - sex chromosomes.
• Such a karyotype remains unchanged throughout life.
• The female sex in humans is homogametic, that is, it has two X chromosomes.
• Male - heterogametic, has an X and a Y chromosome
 EnR- Describe the effect of the following chromosomal mutations on a Biochemistry Mutations
B-017 segment of DNA: point mutation, frameshift mutation, deletion,
insertion, inversion, Robertsonian Translocation, and mosaicism.

MUTATIONS
• Mutation refers to Any change in the DNA sequence of a cell.
• Mutations may be caused by mistakes during cell division, or they may be caused by exposure to
DNA-damaging agents

Different Kinds of Mutations:

Point Mutation Substitution of a single nucleotide base by a different base.
Sickle cell anemia is the result of a point mutation in codon 6 of the b-globin gene resulting in the
substitution of amino acid glutamic acid by valine.
Deletion Deletion of one or more base pairs alters the reading frame of the DNA strand.
Deletion of one codon causes Cystic Fibrosis.
Insertion Insertion of one or more base pairs alters the reading frame of the DNA strand.
Insertion of segment in the factor VIII gene is the most common cause of mutation in hemophiliac
Frameshift mutation If one or two nucleotides are either deleted from or added to the coding region of a message
sequence, a frame-shift mutation occurs and the reading frame is altered. This can result in a
product with a radically different amino acid sequence, or a truncated product due to the
creation of a termination codon
For example, cystic fibrosis (CF), a hereditary disease that primarily affects the pulmonary and
digestive systems, is most commonly caused by the deletion of three nucleotides from the coding
region of a gene, resulting in the loss of phenylalanine at the 508th position
Inversion A portion of the chromosome has broken off, turned upside down, and reattached, therefore
genetic material is inverted.
An inversion is paracentric if the inverted segment is on the long arm or the short arm
The inversion is pericentric if breaks occur on both the short arm and the long arm
Translocation When a part of one chromosome is transferred to another chromosome Translocations are
indicated by t
Centric fusion type or The breaks occur close to the centromere, affecting the short arms of both chromosomes
Robertsonian Transfer of the chromosome leads to one very large and one extremely small chromosome
translocation The short fragments are lost, and the carrier has 45 chromosomes
Mosaicism A person or a tissue that contains two or more types of genetically different cells.
Mosaicism is caused by an error in cell division very early in the development of the unborn baby.
Example:
Mosaic Down Syndrome
 EnR-B- Discuss the concept of central dogma from gene to Biochemistry Central dogma
018 protein (replication, transcription, and translation) (Overview)

CENTRAL DOGMA:
Central dogma refers to three important steps of protein synthesis

1- Replication: It is forming a copy of DNA. DNA replication is the process by which the genome's
DNA is copied into cells. Before a cell divides, it must first copy (or replicate) its entire genome
so that each resulting daughter cell ends up with its complete genome. A double-stranded DNA
molecule is copied to produce two identical DNA molecules.

2- Transcription: It is forming RNA from DNA. Transcription is the process by which the information
in a strand of DNA is copied into a new molecule of messenger RNA (mRNA).

3- Translation: It forms protein from RNA. In this process, the ribosome translates the mRNA
produced from DNA into a chain of specific amino acids. This chain of amino acids leads to
protein synthesis.
 EnR-B- Discuss the gene expression, especially the Lac operon Biochemistry Gene
019 and Tryptophan operon Expression

GENE EXPRESSION
GENE EXPRESSION
• Gene expression refers to the multistep process in which the double-stranded DNA molecule is
copied to produce two identical DNA molecules. hat ultimately results in the production of a
functional gene product, either ribonucleic acid (RNA) or protein

LAC OPERON
• The lactose (lac) operon contains the genes that code for three proteins involved in the
catabolism of the disaccharide, lactose: The lacZ gene codes for β-galactosidase, which
hydrolyzes lactose to galactose and glucose; the lacY gene, which codes for a permease that
facilitates the movement of lactose into the cell; and the lacA gene that codes for
thiogalactoside transacetylase whose exact physiologic function is unknown.
• All of these proteins are produced when lactose is available to the cell but glucose is not.
• The regulatory portion of the operon is upstream of the three structural genes, and consists of
the promoter (P) region where RNA polymerase binds, and two additional sites, the operator (O)
site and the CAP site, where regulatory proteins bind.
• The lacZ, lacY, and lacA genes are expressed only when the O site is empty, and the CAP site is
bound by a complex of cyclic adenosine monophosphate and the catabolite gene activator
protein or CAP (sometimes called the cAMP regulatory protein or CRP, A regulatory gene, the
lacI gene, codes for the repressor protein (a trans-acting factor) that binds to the operator site.

When glucose is the only sugar available:

In this case, the lac operon is repressed (turned off)

Repression is mediated by the repressor protein binding via a

helix-turn-helix motif to the operator site, which is
downstream of the promoter region

Binding of the repressor interferes with the progress of RNA

polymerase, and blocks transcription of the structural genes.
When only lactose is available:

In this case, the lac operon is induced (maximally expressed or turned on)

A small amount of lactose is converted to an isomer, allolactose

This compound is an inducer that binds to the repressor protein, changing its
conformation so that it can no longer bind to the operator

In the absence of glucose, adenylyl cyclase is active, and sufficient quantities of

cAMP are made and bind to the CAP protein

The cAMP–CAP trans-acting complex binds to the CAP-binding site, causing RNA
polymerase to more efficiently initiate transcription at the promoter site

When both glucose and lactose are available:

In this case, transcription of the lac operon is negligible, even if lactose is

present at a high concentration

Adenylyl cyclase is deactivated in the presence of glucose, a process

known as catabolite repression

so no cAMP–CAP complex forms and the CAP-binding site remains

empty

RNA polymerase is, therefore, unable to effectively initiate transcription,

even though the repressor may not be bound to the operator region

Consequently, the three structural genes are not expressed

TRYPTOPHAN OPERON
• The tryptophan (trp) operon codes for five proteins that are required for the synthesis of the
amino acid, tryptophan.
• As with the lac operon, the trp operon is subject to both positive and negative control
• Negative control includes trp itself binding to the repressor protein and facilitating the binding
of the repressor to the operator.
• Repression by trp is not always complete, however, and so unlike the lac operon, the trp operon
is also regulated by a process known as attenuation.
• With attenuation, transcription is initiated but is terminated well before completion
• If trp is plentiful, transcription initiation that escaped repression by trp is attenuated (stopped)
by the formation at the 5’-end of the mRNA of a hairpin (stem-loop) structure
 EnR-B- Discuss the regulation of eukaryotic gene expression with Biochemistry Gene
020 special emphasis on iron metabolism and RNA interference Expression

REGULATION OF EUKARYOTIC GENE EXPRESSION

• The higher degree of complexity of eukaryotic genomes, as well as the presence of a nuclear
membrane, necessitates a wider range of regulatory processes.
• As with the prokaryotes, the primary site of regulation is at the level of transcription.
• Again, the theme of trans-acting molecules binding to cis-acting elements is seen.
• Operons, however, are not found in eukaryotes, which must use alternative strategies to solve
the problem of how to coordinately regulate all the genes required for a specific response.
• In eukaryotes, gene expression is also regulated at multiple levels other than transcription.

Trans acting regulatory element

• Specific transcription factors are trans-acting DNA-binding proteins that function as

transcriptional activators.
• They have at least two binding domains: the DNA-binding domain and the transcription
activation domain.
• The transcription activation domain allows the binding of other proteins, such as co-activators
(for example, histone acetyltransferases, or HATs)
• These facilitate the formation of the transcription initiation complex at the promoter and thus
activate transcription

Cis-acting regulatory elements

• The need to coordinately regulate a group of genes to effect a particular response is of key
importance in multicellular organisms including humans.
• An underlying theme occurs repeatedly: A protein binds to a regulatory element for each of the
genes in the group and coordinately affects the expression of those genes, even if they are on
different chromosomes.

Iron metabolism:
 Transferrin is a plasma protein that transports iron

Transferrin binds to cell-surface receptors (transferrin receptors, or TfRs) that are internalized and provide target
cells with iron

The mRNA for the TfR has cis-acting iron-responsive elements (IREs) at its 3’-end

The IREs have a stem–loop structure that can be bound by trans-acting iron regulatory proteins

When the iron concentration in the cell is low, the IRPs bind to the IREs and stabilize the mRNA for TfR, allowing TfR
synthesis. When intracellular iron levels are high, the IRPs preferentially bind iron instead of IREs

The lack of IRPs bound to the mRNA hastens its degradation, resulting in decreased TfR synthesis

When iron levels in the cell are low, IRPs bind the IREs and prevent the use of the mRNA

thus, less iron is able to be stored, allowing more iron to be transported to cells

As iron accumulates in the cell, however, the IRP protein becomes iron–bound, and loses its affinity for the IRE on
mRNA, resulting in production of apoferritin molecules available to store the excess iron.

RNA interference (RNAi):

• RNAi is a gene silencing mechanism through decreased expression of mRNA, either by

repression of translation or by increased degradation.
• It is thought to play a key role in such fundamental processes as cell proliferation,
differentiation, and apoptosis.
• RNAi is mediated by short (~22 bp), noncoding RNAs called microRNAs (miRNAs), which arise
from far longer, genomically encoded nuclear transcripts that are partially processed in the
nucleus and then transported to the cytoplasm.
• There, an endonuclease (Dicer) completes the processing and generates short, double-stranded
(ds) miRNA.
• A single strand (the guide or antisense strand) of the ds miRNA associates with a cytosolic
protein complex known as the RNA-induced silencing complex, or RISC.
• The guide strand hybridizes with a complementary sequence on a full-length target mRNA,
bringing RISC to the mRNA.
• This can result in repression of translation of the mRNA or its degradation by an endonuclease
(Argonaute/Ago/Slicer) of the RISC.
 • The extent of complementarity appears to be the determining factor.
• RNAi can also be triggered by ds short interfering RNAs (siRNAs) introduced into a cell from
exogenous sources

RNAi therapy:

• Modulation of gene expression by providing siRNA to trigger RNAi has enormous therapeutic
potential.
• The first clinical trial of RNAi-based therapy involved patients with age-related macular
degeneration (AMD), the leading form of adult blindness in the world.
• AMD is triggered by overproduction of vascular endothelial growth factor (VEGF).
• In patients with AMD, too much VEGF leads to the sprouting of excess blood vessels behind the
retina.
• The blood vessels leak, clouding and often entirely destroying vision; hence, AMD is also
referred to as “wet” macular degeneration.
• The siRNA drug—a short dsRNA that specifically targets the mRNA of VEGF and promotes its
degradation—is injected into the eye.
• One siRNA molecule can destroy hundreds of mRNA, resulting in the suppression of thousands
of VEGF proteins, thus preventing damaging angiogenesis in the retina.
 EnR- Discuss the following Recombinant DNA techniques with Biochemistry Techniques
B- reference to their principles, procedures, and application: 1) PCR
021 (Polymerase Chain Reaction) 2) RFLP (Restriction Fragment
Length Polymorphism) 3) Cloning 4) Human Genome Project 5)
Blotting Techniques 6) DNA (Deoxyribose Nucleic Acid)
sequencing

BIOTECHNOLOGY
Polymerase Chain Reaction:
• The polymerase chain reaction (PCR) is a test tube method for amplifying a selected DNA
sequence that does not rely on the biological cloning method
• PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours
• It can amplify the sequence, even when the targeted sequence makes up less than one part in a
million of the initial sample.

Steps:

1. Construction of Primer
2. Denaturation of DNA
3. Annealing of Primer to ssDNA
4. Extension of primers with DNA polymerase

Application:

• Comparison of a normal cloned gene with an uncloned mutant form of the gene
• Detection of low-abundance nucleic acid sequences
• Forensic analysis of DNA samples
• Prenatal diagnosis and carrier detection of cystic fibrosis

Restriction fragment length polymorphism (RFLP)

• A restriction fragment length polymorphism (RFLP) is a genetic variant that can be observed by
cleaving the DNA into fragments (restriction fragments) with a restriction enzyme.
• The length of the restriction fragments is altered if the genetic variant alters the DNA to create
or abolish a site of restriction endonuclease cleavage

Application:

• RFLP can be used to detect human genetic variations

• Tracing chromosomes from parent to offspring
• Prenatal diagnosis
• Direct diagnosis of sickle cell anemia
• Indirect, prenatal diagnosis of phenylketonuria
Blotting:
The most commonly used blotting techniques are listed below

• Southern blotting (for DNA)

• Northern blotting (for RNA)
• Dot blotting (DNA/RNA)
• Western blotting (Protein)

Southern Blotting
• Southern blotting is a technique for identification of DNA molecules

Steps:

1. DNA is extracted from nucleated cells like leukocytes mainly

2. DNA is cleaved with the same restriction enzyme
3. Agarose gel electrophoresis
4. Denature the DNA into single strands and transfer (by a blotting action) to a nitrocellulose
membrane, creating a replica of the gel
5. Hybridization with 32P-labeled probe
6. Expose x-ray film

Applications:

• It is an invaluable method in gene analysis important for the confirmation of DNA cloning
results.
• Forensically applied quantities of DNA (to thieves, rapists, etc.).
• Highly useful for the determination of restriction fragment length polymorphism (RFLP)
associated with a pathological condition

Northern Blotting
• Northern blots are very similar to Southern blots except that the original sample contains a
mixture of mRNA molecules that are separated by electrophoresis, then transferred to a
membrane and hybridized to a radiolabeled probe.
• The bands obtained by autoradiography give a measure of the amount and size of particular
mRNA molecules in the sample.

Western Blotting:
• Western blotting involves the identification of proteins.
• It's very useful to understand the nucleic acid functions, particularly during gene manipulations.
• The technique of Western blotting involves the transfer of electrophoresed protein bands from
polyacrylamide gel to nylon or nitrocellulose membrane.
 • These proteins can be detected by specific protein-ligand interactions.
• Antibodies or lectins are commonly used for this purpose

DNA CLONING:
The introduction of a foreign DNA molecule into a replicating cell permits the cloning or
amplification (that is, the production of many copies) of the DNA.

The process of introducing foreign DNA into a cell is called Transformation for bacteria and yeast
and Transfection for higher eukaryotes.

Types of cloning:

i. Recombinant Cloning
ii. Reproductive cloning
iii. Therapeutic cloning

DNA SEQUENCING:
• Sequencing means finding the order of nucleotides/amino acids in DNA, RNA, and protein
• Two main methods of DNA sequencing are the Maxam and Gilbert method and the Sanger
method
• Maxam and Gilbert's method uses chemical sequencing
• Sanger method uses dideoxynucleotides
No Cortisol and Androgens but increased 17 alpha-hydroxylase deficiency
production of mineralocorticoid
No Cortisol and mineralocorticoid but increased 21 alpha-hydroxylase deficiency
production of androgens

EnR-B-008 (Steroid in infertility)

The primary goal of contraceptive pills is Induce ovulation
Pills used for the control of fertility consist of a synthetic estrogens and synthetic progestins
combination of
The most commonly used synthetic estrogens ethinyl estradiol and mestranol
are
Lack of ovulation caused by hyposecretion of HcG
the pituitary gonadotropic hormones can be
treated by

EnR-B-009 (Nomenclature of genetics)

Location of gene on chromosome Locus
Pairs or series of genes on a chromosome that Allele
determine the hereditary characteristics
A boy inherited only one copy of the allele from Dominant allele
his father for a phenotype and showed that
phenotype. This is an example of
The presence of an abnormal number of Aneuploidy
chromosomes in a cell
A person serving as the starting point for the Proband
genetic study of a family
The human blood group is an example of Co-Dominance
Skin color is an example of Polygenic trait

EnR-B-010 (Genes)
functional units of heredity are Gene
The normal number of chromosomes in 23 Pairs
humans is
Autonomic chromosomes are in total 22 pairs

EnR-B-011 (Mendelian Law)

The two co-existing alleles for each trait of an individual segregate (separate) Law of Segregation
from each other at meiosis, so that each gamete receives only one of the two
alleles
When two contrasting pairs of traits are followed in the same cross, Law of independent
their alleles assort independently into gametes assortment

EnR-B-012 (Patterns of Inheritance)

Huntington’s Disease is an example of Autosomal dominant inheritance
Xeroderma Pigmentosum is an example of Autosomal recessive inheritance
In autosomal inheritance probability of is Equal
spread of disease in males and females is
X-linked diseases are more common in Male
Retinitis pigmentosa is an example of X linked dominant inheritance
Duchenne Muscular dystrophy is an example of X linked recessive inheritance

EnR-B-013 (pedigrees)
In the pedigree, a colored square box Affected Male
represents
In Pedigree, an empty triangle represents Unaffected female
In pedigree, the carrier is represented by Dot inside a normal sign

EnR-B-014 (Mode of Inheritance)

Huntington’s Disease is an example of Autosomal dominant inheritance
Xeroderma Pigmentosum is an example of Autosomal recessive inheritance
In autosomal inheritance probability of is Equal
spread of disease in males and females is
X-linked diseases are more common in Male
Retinitis pigmentosa is an example of X linked dominant inheritance
Duchenne Muscular dystrophy is an example of X linked recessive inheritance

EnR-B-015 (Chromosomal Abnormalities)

Trisomy 21 is Down syndrome
The most common chromosomal disorder is Down Syndrome
A disease in which there is an extra copy of the Down syndrome
21st chromosome is called
Klinefelter’s syndrome results from the nondisjunction of sex chromosomes during
meiosis

EnR-B-016 (Karyotypes)
A particular array of chromosomes is called Karyotypes

EnR-B-017 ( Mutation)
Sickle cell anemia occurs as a result of Point mutation
cystic fibrosis (CF), a hereditary disease that 508th position
primarily affects the pulmonary and digestive
systems, is most commonly caused by the deletion
of three nucleotides from the coding region of a
gene, resulting in the loss of phenylalanine at
A person or a tissue that contains two or more types Mosaicism
of genetically different cells
EnR-B-018 ( Central dogma)
Three important steps of Protein synthesis from Central dogma
DNA are called
The process by which the genome's DNA is Replication
copied in cells
process by which the information in a strand of Transcription
DNA is copied into a new molecule of
messenger RNA
The process by which ribosomes form new Translation
protein from the information encoded in mRNA

EnR-B-019 ( Gene expression )

When glucose is the only sugar available Lac operon is repressed
When only lactose is available Lac operon is induced
The tryptophan (trp) operon codes for ______ Five
proteins that are required for the synthesis of
the amino acid, tryptophan
The lactose (lac) operon contains the genes Three
that code for ______ proteins involved in the
catabolism of the disaccharide, lactose

EnR-B-020 (Gene Expression

Mechanism of gene silencing through RNAi
decreased expression of mRNA, either by
repression of translation or by increased
degradation
Regulation of gene expression typically occurs Transcription
at the level of
The mRNA for the TfR has cis-acting iron- 3’-end
responsive elements (IREs) at its
Age-related Muscular Disorder is triggered by vascular endothelial growth factor
overproduction

EnR-B-021 (Techniques)
PCR permits the synthesis of _____ copies of a Millions
specific nucleotide sequence in a few hours
Technique for identification of DNA molecules Southern blotting
Technique for identification of RNA molecules Northern blotting
Technique for identification of protein Western blotting
molecules