9.1: Human Genetics - Method & Application

Meaning & Scope of Human Genetics

Introduction – study how genes among humans transmit into traits as well as the pattern of inheritance from parents to child.

Definition

Study of process of inheritance.
Scientific study of variation & heredity in human being.

Aims

Principle of inheritance
Nature of inheritance
Variance within the population taxonomy
Application for human welfare

Background

George Mendel, 1866 – experiment on pea plant gave 4 principles → Law of assortment, law of segregation, law of dominance, one gene – one character
1906 – Bateson coined the term from early recorded history of physical traits transferred from parents to children. No scientific proof.
In recent time, extent of genetics has been increasing, Neet & Scherll (1954) – study of trait linked with particular gene – physiological genetics.

Branches

Classical / Mendelian Genetics – Study about rules of inheritance
Molecular Genetics – Examines structure, organisation, transmission & functions of genes ; how it determine traits (phenotypes) ex via Protein Synthesise
Cytogenetic – Study of human chromosome, sex chromatin & chromosomal abnormalities & their consequences
Population Genetics – It focuses upon stability & change in gene frequencies in various human population. I.e Nature & extent of distribution of different genes in various human population group b/c of mutation, selection, genetic shit & gene flow
Immunogenetics – Studies how genes control the immune system of a body. Different types of antibodies, histoincombatiblity (HCA)
Biochemical Genetics – genetics controls the secretion of enzyme which control the biological functioning.
Medical Genetics – Transfer of disease.
- Genetic screening
- Genetic counselling
- Genetic therapy

Significance

It helps in understanding interaction of environment & hereditary on Human being with different trait e.g Sickle Cell anaemia (HbS/HbA) → Balanced Polymorphism
Study of disorder
Impact of Consanguineous marriage
Changing environment & increasing radiation → population genetics → Biological evolution (H-W Law)

Conclusion – it is a vast field that studies the human variation & transmission of trait along with physiological function is an inseparable part of Biological anthropology. Indeed a sub disciplines which transferred physical Anthropology to biological anthropology.

Human Genetics : Why Not Discipline ?

Introduction – Mainly Because Human is highly unfavorable for genetic study b/c of the following factors

Social Hindrances – it is impossible to conduct controlled breeding experiments
Number of offspring – is small, rendering statistical generalization & calculations impossible
Human’s lifespan is long – the result can’t be studied for three or four generations
Genetically Heterozygosity – Majority of human are genetically heterozygous for many characters. Therefore, it is difficult to get isogenic strains for genetic experiments.

Conclusion – Hence to understand we pursue Indirect studies. However The Development of newer techniques has helped in understanding the mechanism of inheritance of no. of a character in man more holistically.

Nature & Nurture Debate

Introduction: Within anthropology & other academic disciplines, there is often much debate b/w the respective influences of nature & nurture.

Nature refers to what we are born with and includes our biology, genetics, and similar characteristics.
In contrast, nurture refers to what is learned, through our cultural community and other individuals.

For anthropologists, this debate often centres on the influences of nature and nurture in different physical and cultural elements.

History: The phrase in its modern sense was popularized by the English Victorian polymath Francis Galton, the modern founder of eugenics & behavioural genetics, discussing the influence of heredity & environment on social advancement. Galton was influenced by the book ‘the Origin of Species’ written by Charles Darwin.

Franz Boas on Nature-Nurture debate:

Early Mendelians insisted that all human traits, including broad behavioural patterns, were genetic, rooted in nature.
Boas argued to the contrary, stressing the richness of cultural diversity, which he said was evidence that behavioural differences were rooted in nurture.
He tried to convince the world that environment was the sole contributor to human behaviour but scientific research has shown that to not be true.
In fact, our behaviour is shaped by a combination of genetics of environmental influences.
Neither can function on its’ own, and neither can influence us on its own.

Study of Newman, Freeman and Hozinger

They studied 50 pairs of identical twins reared together, 50 pairs of non-identical twins reared together and 19 pairs of identical twins who were separated very early in life and reared in different homes and 52 regular siblings as control.
The study showed that the identical twins reared together
- showed differences in four physical traits considered namely the height, weight, head length and head breadth. This was considered to be the non-genetic effect due to environment which has given rise to phenotypic differences inspite of identical genotypes.
- The average differences were found to be larger in non-identical twins for each of the four traits considered. The level differences were same for non-identical twins reared apart and ordinary siblings.
The identical twins reared apart showed
- more differences than those reared together.
Concordance discordance could be seen in the twin pairs in respect of many physiological traits and some pathological conditions.

The twin studies on intelligence test behaviour

It shows that modifiability of Q score is under the influence of differences in environment and there is a greater similarity in IQ of identical twins whether reared together or in different homes, than that of non-identical twins reared in the same home.
Differences in the intelligence of non-identical twins are partly due to differences in their environment and partly to hereditary differences.

An individual is the expression of five interacting & interdependent fundamental factors

A system of genetic relations.
A system of development relationship.
Influenced by the maternal uterine environment.
General socio-economic environment.
General physical environment.

The environment provides the opportunities for the development of innate potentialities which are limited by heredity. People owe their differences to genetic and environmental factors. Both these interact so that the degree of manifestation of genetic factors depends on the environment.

Heritability

For obvious reasons most of the information about the analysis of the Nature-Nurture problem in a man comes from twin studies. In order to separate the relative roles of genes & environment for polygenic traits, the idea of heritability has been developed.
Heritability is defined as the proportion of the total phenotypic variance of trait that is caused by additive genetic variance. For ex the heritability of a metric characters express the proportion of the total variance that is attributable to average effects of genes and this is what determines the degree of resemblance between relatives.
Thus in general terms, heritability is a measure of whether the role of genetic factors in determining a given phenotype is large or small.
One method of estimating heritability is by comparison of the ratio of the concordance rate of the condition in monozygotic and dizygotic twin pairs with the incidence of the trait in the general population.

Conclusion: Today, most academics, including anthropologists, recognize that both nature & nurture influences affect the human beings and their culture. Therefore, the nature versus nurture debate can be considered a false dichotomy, but it still has an impact on research today. However, now most disagreement between academics is regarding the extent of nature or nurture influences.

Genetic Principles

Are the rule or standards governing the biological phenomena of heredity, the transmission of characteristics from parents to offspring via information encoded biochemically using DNA, in units called genes.

As all genetic observations can’t be explained & predicted by Mendelian genetics. Hence scientists have developed various methods to study these genetic principles. Some of these are following

Pedigree Analysis

Introduction – First suggested by Galton & he defined it as – “Pedigree analysis is the study of pattern of inheritance of traits which show regular transmission from generation to generation in a family, with help of certain symbols & methods.

Feature of Pedigree Analysis

A pedigree chart display a family tree, & help to analyse the pattern of inheritance of a particular trait across the family.
Pedigrees chart are based on phenotype & not on genotype
- B/c For genotype, molecular studies are required
Represented by certain symbols c/a Pedigree Symbols
- Heterozygotes or carriers of gene are designed by a black dot in the middle of the symbol or by coloring half the symbol black.
- The person who brought the trait to the geneticist’s attention is c/l Proband, or Propositus if male, and Proposita if female or index case.
- Males are placed on the left & females are placed on the right.

Principal of Pedigree Analysis ; it is done on the basis of Mendelian principles

Law of dominance
Law of recessive
Law of segregation
One gene – One character
Law of equivalence
Law of independent Assortment

Importance / Use / Singnificance / Advantage

Helps in determine genotypes, identify phenotypes
Transmission of pattern of a trait in the next generation. ; also helps to determine the mode of inheritance of trait, whether it is dominant recessive, autosomal or sex linked
Studying Mendelian laws & exception to it –
Useful tool in genetic counseling, which is intended to avoid/reduce incidence of unifactoral disease.
- Pedigree shows, if the character is dominant – couple should go for abortion
- In case of carrier parent, possiblity of having normal child (carrier) can be worked out & also that of having defective grandchild (if wife of carrier child is carrier)
Paternity dispute
It provide information on allelism & linkage.
Since, critically informal mating can’t be designed experimentally, due to factors above . Pedigree is best mean to study inheritance of such disorders.
Since we can’t go forward, but we can go backward i.e Gather info of all the existing members of the family under study & also previous generations & build up a pedigree chart (a family tree of sort)
Analyse the source, mechanism & type of genetic disorders
For research – provide valuable source of information for observing prevalence rate of diff types of genetic disorder.

Basic vocabulary (for Understaffing Purpose)

Genotype: the specific alleles of an organism. (AS, Aa, etc.)
Phenotype: the observable characteristics or traits of an organism.
Dominant allele: an allele that has the same effect on the phenotype whether it is present in the homozygous or heterozygous state.
Recessive allele: an allele that only has an effect on the phenotype when present in the homozygous state.
Co-dominant alleles: pairs of alleles that both affect the phenotype when present in a heterozygote.
Homozygous: having two identical alleles of a gene. (RR or rr)
Heterozygous: having two different alleles of a gene. (Rr)
Carrier: an individual that has one copy of a recessive allele that causes a genetic disease in individuals that are homozygous for this allele. (Ex. Sickle cell – AS or Aa)
Criss Cross Inheritance: If a gene travelled from father to daughter to son. The character from maternal grand father to grandson via his mother.

Key points for Analysis (Understanding Purpose)

An unaffected individual can’t have any alleles of dominant trait
An unaffected individual can be carrier of a recessive trait
When a trait is X-linked, a single recessive allele is sufficient for a male to be affected
A father transmits his allele of X-linked genes to his daughter, but not his sons. A mother transits an allele of X-linked genes to both her daughters & her sons.

In case of Autosomal gene
- equally affects both males and females.
In case of Sex linked gene
- Affects the males as they are hemizygous.
- The gene shows criss-cross inheritance i.e., the gene from the father is transferred to the grandson through the daughters.
- In the case of a sex-linked dominant gene, more females are affected than males.
- Never transferred from father to son.
In case of dominant genes
- One or both the parents have the disorder.
- It expresses itself in every generation.
- The disorder is common in the pedigree.
- The genotype is either homozygous (BB) or heterozygous (Bb).
- It affects one-half of the children.
In case of recessive genes
- Neither of the parents may have the disorder.
- The disorder is rare in the pedigree.
- Both parents are either heterozygous or homozygous recessive.
- The disorder skips generations.
- The genotype is always homozygous (bb).
- Affected offspring are born to unaffected parents.
In the case of Holandric (Y linked Genes)
- Affects the males only.
- Father transfers it to son.
- It never skips generations.
In case of Cytoplasmic genes
- Gene is inherited from mother.
- Affected mother transfers the gene to all its offspring.

Conclusion – Due to its relevance, Pedigree analysis finds huge application in genetic counselling & gene therapy.

Monogenic & Polygenic Inheritance

Monogenic Inheritance

refers to inheritance patterns resulting from a single gene/factor. It is based on Mendel’s Law of Segregation. The different patterns of such inheritance are –

Autosomal Inheritance – is a pattern of inheritance in which the transmission of the traits depends on the presence or absence of certain alleles on the autosomes. They pattern may be
- Autosomal dominant Trait Inheritance e.g polydactyl
- Autosomal Recessive Trait Inheritance e.g Cystic fibrosis
Sex Linked Inheritance – is a pattern of inheritance in which the transmission of traits depends upon the presence or absence of certain alleles on the sex chromosomes.
- X-Linked Dominant Trait Inheritance e.g Rickets
- X-linked Recessive Trait Inheritance e.g Haemophilia
- Y-Linked Inheritance (Holandric) e.g Hairy ears

Polygenic Inheritance

Intro – There are several quantitative traits like height & skin colour that display continuous variations & can’t be simply Mendelian inheritance.

To a/c for such traits R.A Fisher proposed the Multiple Factor Hypothesis. It postulates that there are traits whose inheritance is determined by multiple factors both environmental & genetics. This si known as Polygenic / quantitative inheritance.

It explains why offspring of phenotypically different parents mostly expresses an intermediate phenotypes.
For ex – Mating b/w homozygous tail & homozygous dwarf parent will have heterozygous medium tall progeny.
Conditions attributed to polygenic inheritance includes diabetes, cancer, obesity etc

Types of Pedigrees Illustrating Inheritance

Introduction– Depends upon nature of the gene causing the character under study. Pedigree can be of different types. A gene can be Autosomal Dominant, Autosomal Recessive, Sex Chromosomal Dominant And Sex Chromosomal Recessive

Used in study of inherited diseases rather than normal traits b/c there are better documented pedigree of disease & abnormal conditions.

Autosomal Dominance Trait Inheritance

The Pedigree Pattern includes the Following Characteristics

Present in almost all generations (No skipping of generation i.e Dominant)
It equally affects both males and females. (i.e. equal frequency in male & female)
The Character thus show vertical pattern of inheritance
Every affected person has an affected parent
- Father couldn’t have passed the condition to his son if determined by X linked gene.
- Affected man passes it to his daughter, hence not Y linked
- if X linked produced all affected daughters
Two unaffected parents will not have affected children
If one generation doesn’t express the trait, it is lost forever

Example – Conditions (Trait & Disease) which are caused by this

Huntington’s Diseases (Progressive degeneration of nervous system )
Achondroplasia (Short limbed Dwarfism)
Brachydactyly (Short fingers)
Polydactyly (Extra Fingers)
Porphyria (Skin Lesions due to exposure to sunlight)
Osteogenesis Imperfecta (Brittle bone)
Creutzfeldt Jakob Disease (Senile dementia, Myotonic dystrophy )
Nail patella Syndrome (Abnormal Knees & nails)
Cold urticaria

Autosomal Recessive Trait Inheritance

The Pedigree Pattern includes the Following Characteristics

Thought to be present in equal frequency in both the sexes
Skip generation due to masking of phenotypes
Show horizontal pattern of distribution
Normal parents with affected children, → parents are heterozygous ; & are most likely to to related to each other than parents of normal children.
- Result of consanguineous marriage
- Rarer the disease, the greater frequency of parental consanguinity
On an average 1/4 children will be affected, 1/2 will be heterozygotes and 1/4 will be normal
All children of an affected person will be phenotypically normal heterozygous
All Children of two affected persons will be affected
Presence of affected daughter whoes father & mother are normal again indicates that the gene is not sex linked but autosomal
Could not be dominant as parents are normal

Example – Conditions includes

Cystic Fibrosis (formation of thick mucous in lungs)
Sickle Cell Anemia
Albinism (Absence of melanin)
Phenylketonuria
Alkaptonuria (Black Urine)

X- Linked Dominant Trait Inheritance

100% linkage of affected daughters from an affected father suggests X-Linked Dominance

The Pedigree Pattern Includes the following Characteristics

The nature of inheritance depend upon whether the gene is inherited from father or mother side.
- if from father side, The character will be inheritated by daughters only
  - b/c father has one X which is always given only to daughters.
- if from the mother side, both the boys & girls have equal chances to inherit.
Condition appears in every generation,
Have sex bias.

Example of Condition

Vitamin D resistant rickets
Blood group Xg are X dominant

X – Linked Recessive Trait Inheritance

100% linkage of affected sons from an affected mother suggests X-Linked recessive

Features of Pedigree

Males(Nearly all) are more affected it b/c
- for Female to be affected need to be homozygous as two X chromosomes are present but in case of male only one X chromosome so affected easily
- presence of double dose of such gene in the female zygote is lethal hence they rarely develop.
if the trait is transmitted through the heterozygous mother, she is usually phenotypically normal
An affected male never transmits the trait to his sons.
All daughters of an affected male will be carriers
The carrier female transmits the trait to 1/2 of her sons.

Example : Conditions linked

Hemophilia (faliure of blood to clot upon bleeding)
Red & green color blindness (failure to identify red & green from other color etc)
G-6PD deficiency

Y-Linked Inheritance (Holandric)

Features of Pedigree

implies that only males are affected & trait is transmitted to all his son
Trait never transmitted to daughters

Example

Hairy ears,
Webbed toes

Conclusion – Thus, pedigree analysis can only present a broad indications, the advanced methods in genetics like karyotypic study & DNA Studies have to be conducted to support the hereditary studies.

Family Study Method

Introduction – Family study methods is one of the simples methods to study genetic principles. It uses pedigree analysis to study them. It throws light on the genetic make-up of members of the family.

Principle

A parent passes half of its genes to an offspring hence a parent and offspring has half of the gene common. This half is k/n as Coefficient of Relationship b/w parent & the offspring.
Such coefficient of relationship means that in a totally additive genetic system, an individual is like half of its parent a quarter like its grand parent, a quater like his uncel/maternal aunt & an eight like a first cousiin
- If one sib inherits a gene, there is ½ chance that the same gene willl be inherited by another sib.
- In the next generation, the grand children also have ½ chance of receiving a gene from parents. Hence liklihood of gene transmitted from grand parent to grand children i.e Coefficient of relationship b/w grand parent & grand children = ½ ×½ = ¼
- The same cofficient of relationship exists b/w uncle/aunt & nephew/niece.
- The coefficient of relationship b/w first cousins is ½ ×½ ×½ = ⅛

Method

Data collected at random or from families which have at least one affected child.
Data so collected is compated with standard data based on type of mating & suspected mode of inheritance.

Significance / Uses

To find out whether a disorders is genetic in origin
For diagnosis of some disease ; about its natural history & variation in expression
For the purpose of genetic counselling
Studing inheritance of factors having additive basis, environment component & dominant factors. for ex in a study inheritance of blood pressure, it has been found that
- 16 % of BP variation in population is of environmental factors
- 48 % additive gentic factor
- 36 % dominance
It helps to study the inheritance of certain traits

Conclusion – A comprehensive family history is important in the analysis of any disorder. Acc to Childs (1982), “To fail to take a good family history is bad medicine & some body will be a criminal negligence.” So this method is great significance for geneticists to clinicians.

Twin & Methods of Diagnosis of Zygosity

Definition of Twin – Twin are defined as two offspring born at the same time from the same mother.

Types of Twins In 1874, Camille Dereste gave distinction b/w two types of twins –

Monozygotic Twins –
- They originate from a single zygote
- Zygote splits in the early state & develops into two genetically identical individuals b/c they both split from same zygote
- Hence the MZ twins are also c/a Identical Twins & are of same sex
- It show no effect of mother’ age
Dizygotic Twins –
- Result from fertilization of two eggs by two sperms
- The relations b/w DZ twins are no different form that of siblings
- Hence, DZ twins are also called Fraternal Twins & may be of Different Sex
- They are more frequent in old mothers

Diagnosis of Zygosity – It is the method to know whether the twin pair is born from same zygote or different zygote

The diagnosis is extremely significant when the twins are of same sex & similar looks
Absolute diagnosis is difficult : no single method by itself will be authentic diagnosis.

Methods of Diagnosis

Placental Method
- In the uterus of the mother, the developing zygote is surrounded by three extra embryonic membranes/layers – chorion, amnion, Yolk Sac & allantoin

Chorion & Amnion are used for determination of zygosity.
Placental Conditions vary in DZ & MZ twins ; condition can be
- Dizygotic – Dichorionic, Diaminoitc
- Monozygotic – Monochorionic, Diaminoitic
- Monozygotic – Monochorionic, Monoaminotic
Similarity Method
- In this method, geneticists use traits which show individual variations like tissue antibodies.
- In order to diagnose the zygosity a small skin graft is made
- In this case, only MZ twin can accept the skin from its partner
Statistical Method(Weinberg Differential Equation)
- In a random mating – P stands for frequency of male children & q stands for female children; born in a population.
- Since twins fo same sex are monozygous twins & dizygotic twins may be of same or opposite sex.

So all dizygotic twins can be written as p²+2pq+q²=1

(All Unlike Sex dizygotic twins)/ (all Dizygotic Twins) = (2pq)/(p²+2pq+q²)

(All Unlike Sex dizygotic twins)/ (all Dizygotic Twins) = (2pq)/(1)

All Dizygotic Twins = All Unlike Sex dizygotic Twins ÷ 2pq

Monozygotic Twins = (Total Twins – All dizygotic Twins)

Limitations – It can’t indenify who are monozygotic twins.
DNA Fingerprinting& VNTR
- It si a unique tech. to distinguish one person from another distinctively.
- Base : Every individual has unique constituents of DNA. It is done with the indentification of short nucleotide repeats (SNRs) also known as VNTR (Variable number tandem repeats) which are heritable.
- The VNTS of two persons may be of equal lenght & of equal sequence at certain site but definitely varied at other sites which ensures uniqueness.
- In case of Identical twins, VNTR will show similarity at all sites. On the other hand for dizygotic twins there may be some similarities but definitely there will be differences in terms of VNTR at some sites.
- Hence DNA Fingerprinting can help in determining zygosity absolutely.

Twin Study Method

Introduction – Twin study method is a mechanism through which geneticists can ascertain whether a particular genetic trait has a strong genetic influence in its expression or a strong environmental influence.

Source of Genetic Variation

Genetic variation
Environmental Variation
Interaction b/w Genetics & Environment

Genetic & Environmental Impact on twins

MZ twins arises from same zygote – Genetic Homogeneity
- Any observed difference in their phenotypes – purely environmental in origin
DZ twins by contrast differ b/c of both genetics & environment for a character

Concordance & Discordance

Concordance – if both the members of the twin pair possess a character or both are free from it.
Discordance – if only one member possesses a trait

The extent of concordance or discordance is measure of relative roles of heredity & environment

More equal concordance & discordance ratio b/w the identical & fraternal group would signify less emphasis on heredity & more emphasis on environment in the determination of trait.

Heritability Estmates

Heritability(H) is the proportion of variation that can be attributed to genetic factors. An estimate of degree to which a particular character is genetically influenced – Heritability Estimate

Heritability Estimates are generally based on comparison of concordance rates of Mz & DZ twins
Factor responsible for problems in estimation of heritability
- Humans are not experimental animals
- Phenotypic variation doesn’t just depend upon heredity & environment alone but also the interaction b/w the two, which is difficult to conserve & also quantify.

There are two types of twin methods to estimate heritability

Concordance Discordance Study : H/E based on variance b/w MZ & DZ twins
- comparison of MZ & DZ twins for concordance & discordance. & their rate indicates within the pair variation for MZ & DZ twins
- Once the within the pair variation is known, Coefficient of Correlation can be calculated

Coefficient of Correlation = (V_DZ – V_MZ) / V_DZ

V_DZ = Within-the-pair variation of DZ twins

V_MZ = Within-the-pair variation of MZ twins

For a character with strong genetic tendency, the value of V_MZ will be extremely low, compared to value of V_DZ
- Hence, the value of Coefficient of Correlation will be reaching one
If a particular character has strong environmental influence, the value of V_MZ would be high & may be same as the value of V_DZ
- Hence the coefficient of correlation- approaches Zero
By the values of genetic & environmental component of all characters, we can calculate heritability using the formula

H = V_G/ (V_G + V_E) = V_G / V_T

The method is used for finding out the heritability of discrete characters.
These findings can be confirmed with the help of chi-Square test

Rearing Study : H/E based on MZ twins reared together & apart
- This is based on the assumption that since the twins are genetically similar, any variation among them is purely environmental in susceptibility

Coefficient of Correlation = (V_A – V_T) / V_T

V_A = Variation in MZ twins reared apart

V_T = Variation in MZ twins reared together

In case of strong environmental influence, the value of V_A would be greater & value of V_T would be small &
- hence the coefficient of correlation would be approaching one
it there is strong genetic influence, the value of V_T & V_A would be similar &
- hence the value approaches 0
Rearing studies are performed to carry out / to find out heritability of continuous traits such as height, weight, intelligence.

Case Study

by Water Jablonski in 1922 – Investigation of contribution of heredity to refraction to human eyes. Examined 52 twin pairs – compared size diff of within-pair differences b/w identical & nonidentical twins
By Thomas J.Bouchard, Jr. & colleagues at university of Minnesota – study included twins separated since birth & raised by different families (adoption studies), and so assumed that similarities, if found any, must be those that are heavily influenced by a person’s genetic heritage.

Uses of Twin Study

to estimate the role of Hereditary & environment in the formation of character & also international b/w two which is referred as Nature-Nurture Interaction b/w heredity & environment
Useful for drug testing traits in the treatment of disease – therapeutic trails
To study m/c of transmission of genetic disease in the population.
Twin study is very important in understanding behavioural genetics

Viability of Twin Method

Assumes presence of genetic & environmental components without actually specifying them
Doesn’t recognise the nature, locality & behavior of genes nor does it recognise the physical, Chemical & biological components of environment
Fails to tell about the actual gene concerned, or their pattern of inheriance
Can’t be used in estimating the complex characters like intelligence,
- B/c they are caused not either by heredity nor environment but by complex interaction b/w them
Assumes that MZ twins are exactly similar, but evidences shows that they too differ in characters like size & vigor
Can’t be used in estimating the intra-uterine environment– difference here might lead to low concordance in MZ twins
Twin method can’t be applied to analyze like congenital malformations.
A majority of environmental factors are still being neglected. Hence the change brought about by these factors may be falsely assumed to be genetic.
Sampling of the environment of twins-need not be the environment of population as a whole
Inter population variations-not accounted for.
- for ex. A particular trait in one population may differ due to environment but it can differ due to genetics in another population.
Twinning is a rare phenomenon-generalizations questionable in their objectivity

Conclusion – thus twin study has huge relevance in genetics, especially in understanding & draw reasoning for the long standing concept of the role of nature & nurture in the development of traits.

To overcome some of the issues, Co-Twin method was developed as correction to twin method

Co-Twin Method

Introduction – it is one of the method of separation of hereditary & environmental factors in development of trait.

Concept of Monozygotic & Dizygotic Twins

Co-Twin Method

In Co-Twin method, the identical twins along with its co-twin & the fraternal twin along with its co-twin are investigated & compared.
This is different from twin studies, wherein each pair of twins is studied as a combined unit, & then compared.
Such investigation when done under controlled condition, is called co-twin control method.
But disparities exist b/w the conclusions of the twin & co-twin method when compared for same behavior. b/c
- Cotwin would be counted twice for the concordance rate whereas they should have been counted by once.

Limitation

Limitations of this method are similar to that of twin method,- it doesn’t specify the exact genetic & env component.
It also fails to pinpoint actual genes concerned, or their pattern of transmission.

Examples – Impact of psychological Exercise on intelligence ; after the training the – performance of trained twin improved, but not that of their co-twins.

Use of Co-Twin method

Separating influence of heredity & environment in development of a trait
Detemining effect of environment in development of trait
Indentifying importance of heredity in detemination of a trait
Study of mental traits
Study of Mendelian inheritance in man.
used to understand no. of abnormal behaviors, especially schizophrenia
Large simple size sometime not possible

Foster Child Method

Introduction – It is another method used in the analysis of nature-nurture influence in the development of traits, especially for analysis of intelligence.

Separating respective influence of heredity & environment in the development of trait.
Ex. Chicago school of studies of foster Child.

Technique

Children are selected at random.
Placed in different homes classified as good, average & poor
Since the group of children selected is random, the genetic factor of the trait selected is assumed to be equally distributed in them
After lapse of time, they are tested on different intelligence scales
If intelligence has environmental component, children placed in good homes should score better than those in average & poor ones.

Requirements -Osborene -1951 gave following required while using this tech

Foster children must be placed in the adoptive homes quite early-uninfluenced by environmental of original home
No selective placement
Adequate samples of children from various social levels
Must be from one population to eliminate variations due to ethnicity & race

Limitation –

This method is not free from biases & subjectivity.
Ethical Issues
Only realises doesn’t specify the environment factor
Complex characters can’t be attributed to be any one environment ; as character like Intelligence, behaviour are result of interaction b/w genes & environment
Majority of environment factor still neglected – falsely attributed to genetics.

Case Studies

Chicago Studies
- selective placement was greatly minimized
- The study revealed that the mean IQ scores of the adopted children were related to the quality of the adoptive homes.
- Such experiment show a noticeable effect of environment on the development of intelligence.
Minnesota Graded Home Study
- Children of managerial class were placed in labor class & vice-versa
- It was found that there is effect of environment on IQ but at the same time it was also found that children of managerial class had more IQ in labor class homes than children of labor class in their own homes.
- This indicated importance of heredity in determination in IQ.’

Conclusion– foster child method is a key tool in behavioural genetics, & it often combined with twin studies to make heritability estimate.

Cytogenetic method

Introduction – Cytogenesis is the study of chromosomes & the related disease states caused by abnormal chromosome number &/ or structure. This branch developed in late 1950s & thereafter the development & research in this filed have been tremendous.

study of human chromosome, sex chromatin & chromosomal abnormalities as well as analyse their impact on biological growth & development.
Cytological investigation to genetic material

Methodology

Examining the chromosomal behaviours during mitosis & meiosis as chromosome are carriers of gene i.e functional unit of traits

Process

Normally chromosomes can’t be seen with a light microscope but during cell division they become condensed enough to be easily analysed at 1000X
- best visible during metaphase of mitotic cell division, when they are arranged at the equator.
Variety of tissue type can be used to obtain chromosome preparations. Mainly obtained from the tissues that are actively dividing or are induced to divide in culture b/c chromosome are best visible during metaphase stage & they are exposed to mitotic inhibitors which blocks formation of the spindle & arrests cell division at the metaphase stage. Tissues used to obtain are – Peripheral blood, Bone marrow, Amniotic fluid, products of conception
Method of obtaining chromosome
- sample log-in & initial setup
- Tissue culture (feeding & maintaining cell cultures )
- Addition of a mitotic inhibitor to arrest cells at metaphase
- Stain Chromosome preparation to detect possible numerical & str. changes.
Such chromosome preparations are then subjected to various techniques to identify possible numerically & structural changes.

To visualise the chromosome str & organisation, various techniques such as Karyotyping, analysis of G-banded chromosome, as well as molecular genetics are utilised.

Applications of Cytogenetic Method ;

Determining R/l b/w chromosomal abnormalities & diseases
Mapping genes to particular chromosomes

Various Method to Study of Chromosomes

Traditionally observing chromosome under the microscope. (Mainly morphological characteristic such as – Relative lengths, Arm Ratios & Presence or absence of 2° constructions.
Chromosome Banding Technique
In-Situ Hybridisation with DNA Probes
- Simple Hybridisation
- Fluorescence Hybridisation
Karyotyping / Idiogram
Computer Assisted Chromosome Analysis
- To overcome the error in measuring the chromosome while preparing Karyotype , chromosome curved or overlapping at metaphase plate ,
- Interactive compute assisted image processing systems ;
- Thru these in-situ hybridisation can also be analysed.

Significance of Chromosomal Analysis

it is helpful in identifying many congenital disease & syndromes which are related to chromosomal aberrations for e.g. Down’s , Turner’s Syndrome.
Genetic & point mutation disease can’t be detected.
Also help in study of cellular function
it helped a lot in correlating various human diseases, malformation & deformities with the abnormalities in the no. & str. of chromosomes.
Taxonomical relationships – karyotypes of different groups are sometimes compared & similarities in karyotype are presumed to represent evolutionary relations.
information on evolutionary past

Karyotype Typing

Introduction – Karyotyping is a laboratory technique utilised for the visualisation of chromosome under a microscope. It is one of primary mechanism for the analysis of human chromosomes.

Basis of Karyotyping

The Karyotype of any organism including man is based on size of chromosome, shape of chromosome, position of centromere, lengths of arms, secondary constriction of an individual species, genus or larger grouping.

Meaning of Karyotyping – Karyotype is systematised arrangement in Standardised Format of chromosomes of a single cell prepared either by drawing or by photography in which the pairs of homologes are ordered in a series of decreasing size.

In other words, the arrangement of whole chromosome of a cell is knwon as karyotyping.
chromosomes of single cell can typify the chromosomes of an individual or even a species.

Ideogram– is the diagrammatic representation of a karyotype which maybe based on measurements of chromosomes in several or many cells. This sort of arrangement of chromosomes represents relative morphology of chromosomes.

Procedure of Karyotyping

Blood 🩸 leukocytes are separated from the blood & are stimulated to divide by mitosis in vitriol by adding phytohaemagglutnin.
Blocking of mitosis at metaphase by applying colchicine
Separation of chromosomes – cells are treated with hypotonic saline solution – This swells the cell & provide clarity to chromosome & helps in counting.
Findng tech to increase resolution & staining differet parts of chromosomes
Precise indentification of individual chromosomes
Microphotographs
Individual chromosomes are cut out of microphotographs
Linking of chromosomes with appropriate partners & are arranged in orderly fashion in homologous pairs – this arrangement is called Karyotype.

Characteristics of Human Chromosomes

Depending upon the position of centromere & the relative length of 2 arms, the photograph of chromosome are artificially arranged in the order of descending length in 7 groups from A to G.

Group A – Pairs 1 to 3 : Large metacentric chromosomes
Group B – Pairs 4 to 5 : Large sub metacetric chromosomes
Group C – Pairs 6 to 12 & X ; Medium sub metacentric
Group D – Pairs 13 to 15 : Large acrocentic with satellite
Group E – Pairs 16 to 18
- 16th – Meta centric
- 17th & 18th – Smallsub metacentric
Group F – 19 & 20 : Small metacentric
Group G – 21, 22 & Y ; Short acro-centric chromosomes with satellite
- but Y don’t have satellite

Method of Karyotyping

Banding Method
Flow Cytometry or Fluorescent Activated Cell Sorting
- is a recent method of chromosomal analysis.
- Cells are ruptured, stained with fluorescent dye which selectively stains the chromosome.
- The chromosome are then projected as fine jet through a flow chambers across a laser beam.
- This causes chromosome to fluoresce which is measured by a detector.
- Since the amount of fluorescence depends upon the size of chromosomes, a rapid karyotyping is possible.
Somatic Cell Hybridization
- Hybrids of somatic cells can be produced
- In such hybrid cells, human chromosomes are eliminated as hybrid cells divide, & some of the human chromosome is retained.
- Under such condition, it is possible to study specific proteins produced by chromosome retained.

Significance of Karyotyping –

helps in proper identification & numbering of chromosomes.
Any gross morphological change of abnormality in the shape or sie of ay chromosomes is easily identified.

Chromosomal Banding Technique

Introduction – Developed during 2nd half of 20th century. Allow the identification different regions of the chromosomes that differ morphologically with great degree of accuracy.

Also the chromosomes that posses similar morphological attributes.
Permits us to establish a correlation b/w linkage maps & cytological maps.

Process –

This involves staining the chromosome with fluorescent dyes.
The staining gives different pattern of bands & inter bands i.e stained & unstained regions along the length of chromosome.
The banding pattern of a particular chromosome remain constant for a particular treatment.

Types of Staining & Bands – At present four such banding pattern are known.

Q Banding or Quinacrine Fluorescence Banding
- Dye – Quinacrine / UV light ;
- Fluorescent bands observed on human chromosome by staining with quinacrine mustard & observed under UV Light. I.e fluorescent microscopy
  - Chromatids are stained, except the distal ends
- It produces characteristics bright & dark bands on chromosomes
- The bands are rich in A-T bases, compacted by proteins.
- In their width, brightness & position there Q bands are so unique that individual chromosomes could be identified & Q band Karyotype could be constructed.
G Banding or Giemsa Banding
- Produced by staining with Geimsa stain
- The bands occur on the same locations as the Q bands
- the bands develop due to compaction of chromatin by non histone proteins
- no florescent microscopy required
C Banding or Centromeric Banding
- this method involves harsh pretreatment of chromosomes with dilute acid followed by strong alkali & warm saline treatment prior to giemsa staining
  - b/c of this much of non histone & histone proteins are removed.
- Under such condition, centromeric regions, particularly of chromosome 1,9,16 & Y chromosome are intensely stained
- Statin all Constitutive heterochromatin localised to particular site on the chromosome (ex -Centromere region)
- The C band +ve regions are sites of concentration of satellite or repetitive DNA.
- There has been found individual variations in the pattern of C banding.
R Banding (Reverese Banding)
- involves saline heat treatment prior to Giemsa staining. this is reverse to G method
- The banding pattern develops b/c of differential response of chromosomal proteins to denaturation.
- Acridine orange
- Reverse banding / all areas which lie between Q bands

In recent years – bands detected after treatment with restriction enzyme → RE Band

Significance

The banding patterns of each chromosome is specific & makes it possible to identify each individual chromosome which may differ in individuals.
These structural polymorphisms, called hereromorphism, are inherited in a simple mendelian fashion & therefore can be studied in the transmission of a chromosome from one generation in the next
Comparing chromosome pattern with normal banding pattern, abnormalities in different chromosome can be easily identified.
In some cases bands can also be used as markers .

In Situ Hybridisation (ISH)

Introduction – In addition to Karyotyping, cytogenetic uses more advanced ISH method. There are 2 types of in-site hybridisation :- Simple & Fluorescence Hybridisation. In these probe bids to only those portion of DNA with which it is complementary. Thus helps →

spatial temporal patterns of gene expression

Example – In diagnoses of

chronic myogenic leukaemia
Angelsnow syndrome

Simple In-Situ Hybridisation

In this technique we locate the physical position of a known DNA sequence on a chromosome – helps physical mapping of genes or repeated DNA sequences.

Steps : Procedure

DNA with in the cell is denatured by treating the cells that have been squashed on a cover slip
The squashed cells is incubated in solution of labelled DNA whose position on a chromosome we are interested in knowing. (Labelled DNA probe can be either Radioactively labelled or Biotinylated probe (Utilize colorimetric detection)
Wash the hybridisation mix & observe them under radiography (in case of radioactively labelled) or by staining with Giemsa (for Biotin labelled probes)

Fluorescence In-Situ Hybridisation (FISH)

In it molecules or labelling probe that have affinity to fluorescence Molecules (which will be deposited on it) are used.

the sites thus located with exhibit fluorescence & can be photographed with a fluorescent microscope.

Advantage

Higher resolution, sensitivity & speed
2 or 3 colour can be used on the same slide for simultaneous detection & localization of serval DNA sequences in the same nucleus.
Entire genomes, whole chromosome, chromosome segment or single copy sequences can be highlighted depending upon complexity of probe used
Can also be used for gene mapping in addition to studying structural & numerical changes in chromosomes.

A linkage Map (aka Genetic Map) is a table for a species a experimental population that shows the position of its known genes or genetic markers relative to each other in terms of recombination frequency, rather than a specific physical distance along each chromosome.

Biochemical Methods

Introduction – Biochemical method or techniques are used to determine the activities of the genes within cells. It is the field of Biochemical Genetics, which combines genetics & biochemistry to elucidate the nature of metabolic pathways by determines the activities of genes within cells to analyze the substrates & products of gene controlled reactions

Biochemical genetics is widely used to identify inborn errors of metabolisms. Ex of many discovered are – PhenylketonuriA, Alkaptonuria, Tyrosinosis, Albinism, Goitrous Cretinism, Gout, PKU, Cystinuria, Galactosemia
Biochemical genetics was initiated by English physicin A.E, Garrod in 1909 in his studies of hereditary diseases. He concluded that hereditary diseases are due to the impact of mutant gene which act as a block of metabolic system. Thus “One Mutant Gene – One Metabolic Block.
Like Mendel’s law it was underground until Beadle & Tatum rediscovered it with there “One Gene – One Enzyme” Hypotheses in 1941

Every activity in the human body, including the appearance of a phenotype, is a biochemical activity i.e initiated & controlled by some sort of biochemical reaction at the cellular level. So , biochemical Method determine the role of genetics in life process

A number of biosynthetic steps transform a precursor to end product.
- All the steps constitute a metabolic pathway.
- Each step of biosynthetic pathway is catalyzed by an enzyme
- which is turn is synthesized by the control of a specific gene.
- The relations b/w Gene & Enzyme is given by Beadle & Tatum & is called the One Gene One Enzyme Hypothesis

Important Method For Biochemical Analysis

A no. of special tech. for separation of biochemical substances & thier analysis have been developed.

Separation & Identification of Proteins – following methods are used by specialists to extract proteins
- Chromatography
- Electrophoresis – separation of protein on the basis of their electric charge. when put in electric filed they move towards +ve/-ve poles.
- Gel filtration – separates protein by their molecular weight. The mixture of proteisn is passed thr a column filled with a polysaccrides called sephidex, present in form of beads with inbuilt holes & protein with high molecular mass can’t pass.

After the proteins are separated, their amino acids can be analysed using Western Blotting Techniques (aka Protein Immunoblot). By HGP we have been able to code all the Amino acid. Thus knowing the defective Amino Acid, we’ll be able to identify the defective genotype.

Sequencing DNA – sequenced & analysed by By Maxam Gilbert method – 4 Restriction enzyme cuts DNA at different bases. These 4 sets of DNA each having different length are analysed → represent position of enzyme
Southern, Northern & Western Blotting are used to detect the presence of specific DNA, RNA & protein respectively,
- by first separation them eletrophoretically & then blotting on nitrocellulose filter.
Any correlation to specific gene action can diagnosed & any corrective action can be taken, if possible.

Significance – 4 major areas where research work is being conducted actively

Human Biochemical Error – there are due to mistakes in metabolic processes fro which actually gene mutation in responsible.
Normal & Abnormal Hemoglobins – helped us to clarify r/l b/w gene, protein & disease
Genetic Variability or Drug Action
Bioinformatics & Computation Biology

Biochemical method further enhance the causes & reason of phenotypes. In future with genetic therapy → will be able to overcome.

Biochemical Markers in Red Blood Cells

Introduction – The biochemical markers in red blood cells are of three types

Red Cell Antigens : Blood Group Polymorphisms
- MNSs System
- RH System
- ABO (H) Secretion
Haemoglobin
Enzymes

Red Cell Antigens ; Blood Group Polymorphisms

MNSs System
- discovered by Landsteiner & Levine when they found two human antigenes which they named M & N. if M antigen is found in blood type c/a M blood group type & if N is present it, is c/a N type blood group.
- Sanger & Race discovered another antigen c/a S in 1947. It differs from M & N but it has genetic relation with M & N. it occure among individuals of all three MN blood types.
- There exists a natural antiboy for S. There are two antigens S & s & three blood types, SS, Ss,ss on the basis of two antigen.
- Now MN & Ss systems are considered togther & hence in the combined MNSs system ten Genotypes are expectd these are

RH System
- In 1940, Landsteiner & Wiener discovered Rh factor in the blood of rhesus monkey. if the person posses it then Rh +Ve & who lacks Rh -ve
- This Rh system is quite independent of the ABO blood group & MNSs blood systems.
- It shows the racial singnificance in different population.
  - Among Mongoloids Rh +ve very rare (0.5 to 1.5 %)
  - Among whits Rh +ve (15%)
  - Among Negros Rh -ve 5 to 8% of population
- Erthroblastosis fetails – Mother Rh-ve & foetus Rh +ve by marriage with a Rh +ve male.

ABO(H) Secretion
- ABO antigens occur in many other tissues along with red blood cells.
- Two forms of ABO antigens
  - Alcohal Soluble – found in all individuals & occurs in most body fluids & secreted by all individuals.
  - Water Soluble – some have water soluble form of A,B or H bg specific specific substances specially in their secretions, esepecially the saliva. These individuals are c/a secretors. & others c/a non secretor.
- Two alleles Se & se control the ability to secrete water soluble A,B or H group specific substances.
- The gene for secretion ability Se is dominant over non secretor.
- These alleles are indepedent of those of ABO blood groups.

Haemoglobins

Hb is ferroproteins composed of globin & haem.
In human, there are 4 types of physiological haemoglobins. They are
1. embryonic, (disappears by 3rd month of embryonic life)
2. foetal,(normal hb of intrauterian period & composes more than half of the total hb in newborn & almost completley disappears till age of 2. however less than 1% is retained by adults also.)
3. adult (constitute about 98% of total hb)
4. A2 (constitute 2 % but maybe raised or lowered in certain abnoramal conditions.)
it is very imp. biochemical marker b/c
1. varies with age, especially duting early period of life
2. Mutation has produced several types of haemoglobin variant which are distributed in diiferent wasy among different populations of different regions of the world.
3. Change in gene frequency can be correlated with environmental factors implicating epidermilogical problems.
4. Certain variants produce anaemia of different natures. In some cases there are heterozygous advantage.
Common Hb variants or abnoramal Hb are; HbS, HbE, HbC
- Haemoglobin S(HbS)
  - it produce Sickle cell anaemia(homozygous) (molecular disease) – +ne of peculiar elongated & sickle shaped cell in blood
  - it is the anamal in beta chain.(beta 6 Glu →Val)
  - heterozygous condtion anaemia is milder order.
  - Homozygous show severe clinical symptoms among which are enlarge spleen, Rheumatism, impaired mental function besides aneamia. Morality rate is high
  - gene frequency in India (5 to 30 %)
  - Hetrozyogte advantage : relatively more resistant to the malaria infection in comparision to noramal homozygotes.
- Haemoglobin E(HbE)
  - widely distributed
  - In different part of Southeast Asia an association is found b/w HbE & Anopheles mosquitoes.
- Haemoglobin C(HbC)
  - it is the anamal in beta chain.(beta 6 Dlu →Lys)
  - both hetro & homo are asymtomatic but homo may develop abdominal & joint pains, jaundice, haemorrhagic pnemona & moderate anemia.
- Thalassemia
  - Hereditary disorder of Hb synthesis.
  - Type
    1. beta Thalssemia
      1. T. Major by Homozygous state – severe haemolytic aneamia (RBC destroyed) perspm don’t survive.
      2. T. Minor by Heterozygous state – causes mild anemia
    2. alpha Thalsemia
      - concerns with alpha chain deficiency
  - also c/a Mediterranean anemia due to frequency of occurence there
  - in some zones of endemic malaria thalassemia is very frequent

Enzymes

Glucose-6-phosphate-dehydrogenase (G6PD)
- is a red cell enzyme which noramlly metabolizes glucose in the red blood cells.
- in case of its deficiency : G6PD Deficiency
  - if person treated with cetain antimalarial drug develop haemolytic anemia.
  - found in Africa, Southeast Asia, Indonesian archipelago,Burma, India & some countries bordering Mediterranean sea.
- its deficiency is inherited. It is a X linked trait. Incompletely dominant.

Biochemical Markers in Plasma

Haptoglobin (HP) – is alpha globin. combines with haemoglobin in the plasma.
Transferrin (TF) – is a beta globin. imp funtion – transport oxidized iron to & from the bone marrow & other tissues.
Albumin – serum protein, shows several polyorphic varieties, which have been reported from different populations
other than aboce,there are several serum proteins such as group specific c omponent(GC), Alkaline phosphate, Ceruloplasmin, Gm system etc.

Immunogenetics – Immunological Methods : Scope & Application

Introduction – Immunogenetics is the study of genetics basis of the immune response i.e its is concerned with the inter relation of heredity, disease & the immune system & its components.

History – the Branch truly emerged in the 1980s, with the discovery of genetically determined cellular surface structures, which control immunological reactions.

Principle – Immune Reaction

is based on the principle that immune reaction are the result of complex gene regulation in our WBC. These reaction are depended upon antigen-antibody interaction, which form the basis of the study of immunogenetics.
The ability of our body & cells to resist invasion by foreign objects, be they cellular, viral or chemical is called the Immune reactions
Many proteins are antigenic i.e. when introduced into an organism, they stimulate the production of antibodies. Various antigens exist in blood
There are 5 types of antibodies namely IgA, IgG,IgM,IgE,IgD each made up of 2 heavy chains.
All these antigens are genetically determined & their study constitutes immunogentics
Blood antigens of man include inherited variations & the particular combination of antigens in an individual is almost as unique as fingerprints
It has been found that antibodies elicited by different individuals differ in aminoacidic composition of different chains.

Scope – The focus, of immunogenetics revolves around

Immunoglobulins (Ig) – In placental mammals there are five Ig isotopes k/n as IgA, IgD, IgE & IgM. Antigen – antibody reaction & variations among antibodies are made use of in immunogenetics. Suitable antigen is injected & antibodies are separated & then purified. Thus, their AA components are determined.
Auto-immune Disorders – it IG studies the genetic basis behind the disease like rheumatoid arthritis & Crohn’s disease. Such disease are caused by an extreme reaction of the immune system against the body’s own tissues.
Blood group system & Rh System

Method

In placental mammals there are five Ig isotopes k/n as IgA, IgD, IgE & IgM. Antigen – antibody reaction & variations among antibodies are made use of in immunogenetics.
Some antigen is injected into man & antibodies are separated & purified
- Such antibodies have been studied & their amino acid composition identified
- Is has been found that antibodies elicited by different individual differ in amino acid composition of different chains.
Such antibodies, injected into experimental animal, in which it will behave as antigen.
The antibody produced by the animal – that is antiserum is taken
With this anti sera blood samples can be tested & one will observe how humans differ in genetic variability.
Also, technique used in immunogenetics is immunological method in which different types of antibodies are present in the blood are separated by using electric gradient. Then the level of antibodies present in the blood is compared with normal levels.

Example – ABO Blood group is done using this method ;

first identified by Austrian immunologist Karl Landsteiner in 1901
He found that ABO Incompatibility & clumping of blood when different types are mixed b/c type A individual have anti-B antibody molecules present in their blood & type B people have anti-A-Antibodies.
Type O individual are found to have both anti-A & anti-B antibody molecules.
On the other hand type AB individual have no antibodies to The ABO group of antigens at all.

Application

Immunological techniques are used in blood group determinations, in organ transplants, RH Factor compatibility etc.
Some specific HLAs are associated with human diseases & disease predispositions
Major area of research also includes autoimmune diseases like Diabetes Type 1, Multiple Sclerosis, Rheumatoid Arthritis etc.
Immunological methods can also be used to understand genetic similarities & differences between individuals & between species(evolutionary distance) – anti sera testing.
The genetic basis of Antibody Molecule
Study of str., function & cellular control of immunoglobulins
Immune responsiveness of an individual
Histocompatibility study.
Detection of Immunodeficiency diseases which are hereditary in nature. Like IgA deficiency, Severe combined immune deficiency syndrome, Wiscot Aldrich syndrome etc.
genetifc counselling of parents having traits of hereditary immunodeficiency disorder
Timely tratment of immunodeficieny disoders.

Conclusion – In addition, new studies are being carried out to support stem cell & organ transplantation. Immunogenetics of aging has also emerged as a hot topic, focusing on the importance of genes regulated immunological functions in maintaining human longevity,

Recombinant DNA technology

Introduction – The terms “Recombinant DNA technology, “DNA cloning”, molecular cloning, or “gene cloning” all refer to the same process i.e transfer of a DNA fragment of interest from one organism to a self replicating genetic element such as a bacterial plasmid ; DNA of another species.

Letters-Nucleotide Base, Words-Codons, Sentences-Genes, Language-DNA.’

History – In 1970, biochemists at Stanford University showed that the genetic could indeed be transferred from one organism to another.

Principle – the technology is based upon structural & functional similarities of DNA among all biological beings from viruses to human being. The process of replication, protein synthesis & genetic code are similar among them. B/c of this rDNA tech is possible.

Purpose – The purpose of splicing the gene into the host DNA is the produce many copies of it. & as bacteria reproduce in a very short time & had plasmids, it is possible to make millions of copies of the gene fairly quickly.

rDNA Technology Steps

Generation of desired DNA Fragments
1. Cut the DNA from another organism with restriction enzymes
Obtaining the Vector to Carry the Desired Gene –

A vector is a carrier of foreign DNA molecule which can replicate independently with in host org to reproduce. Can be Plasmid, bacteriophage, cosmids (most commonly used Plasmid

Cut the Bacterial DNA with restriction enzymes to obtain sticky ends. That is identical complementary ends
Remove bacterial DNA(Plasmid)
Recombination of Desired DNA fragment with DNA of Vector
- Combine the cut pieces of DNA together with another enzyme DNA ligase
- This results into rDNA Molecule – it is the DNA that has been cut from one strand of DNA(from one organism) & then inserted into the gap of another piece of DNA that has been broken (in another organism)
Transfer of rDNA to host Organism (Bacteria)
- can be done by combining them in a test tube with CaCl2 as high conc. of calcium ions makes the membranes of the bacteria more porous.
- Viral vectors are also use to inject the rDNA into animal
Reproduce the recombinant bacteria in Culture Medium
- Organism made to multiply ; plasmid with rDNA starts multiplying in host
- The foreign genes will be expressed in the bacteria.
Selection & Harvesting clones containing Relevant DNA Fragment

Advantages

tech. doesn’t requires large scale investment
The formulartory phase for the establishment of this tech. is also not time consuming. therefore it has more access to masses.

Disadvantage

may introduce potential hazards in environment
- if gene responsible for cncer are cloned in Ecoli & they escape from laboratory it may spead cancer.
possibilty of production of pathogens
BT brinjal – possible gentic effects

Application

used for gene mapping & analyzing gene structure & function
diagnosis of genetic disorders (either directly or by linkage with restriction fragment length polymorphisms) of both adults & foetus.
In case of foetus, the tech. has helped in detection of sex of the foetus.
Production of synthetic vaccines like BCG Vaccines
Gene therapy, particularly in gene replacement
Biosynthesis of hormones like Growth hormone & insulin e.g National Dairy Research Institute recently biosynthesised insulin from buffalo milk thr rDNA tech.
Agriculture e.g in Nitrogen Fixation
Prepare DNA probe which help in identifying – DNA of micro org, Mutation or change , & also used in DNA finger printing

Explanation of Few Uses of rDNA technology

Medicinal – It has brought revolution in diagnosis, treatment & prevention of many diseases.
- Diagnosis : Monoclonal antibodies formed by fusion fo tumor causing genes & gene of leukocytes. It is used in
  1. Blood typing
  2. Pregnancy test
  3. Detecting presence of pathogens such as virus & bacteria
  4. It is also useful in early & accurate detection of cancer.
Analysis of Gene Structure
- this tech has enabled us to learn a great deal about human genes, particularly the Beta globin gene region.
- By arranging the fragments generated by various restriction enzymes(restriction mapping). It has been possible to know the genes & their arrangement in this region.
- using appropriate probes , its has been possible to hybridize the probe in site & find out the location of the gene on a particular region of a specific chromosome.
- For ex. using this tech. it has been possible to know that beta globin genes are localized near the centromere of the short arm of chromosome 11.
- Diagnosis of Genetic Disorders in Foetus
  - DNA of foretus is extracted from Chorionic Villi, cleaved with restriction endonulcease & electrophoreded.
  - One of the DNA fragments, 3, contains beta globin gene.
  - A DNA probe for this gene is obtained from reticulocytes by extracting & separating mRNA of beta globin gene & then obtainin copy DNA (cDNA) of beta globin gene using reverse transcriptase & radioactive Phosphorus, P32.
  - This cDNA probe hybridizes with beta globin gene of foetus presen ton fragment B.
  - The probe will not hybridize if foetal beta globin gene is defective.
  - Sickle Cell Gene
    - if foetus contain normal genes, only normal DNA probe will hybridize;
    - In this way, rDNA tech is useful for locating not only normal genes but genes causing genetic disease also.
DNA Probes – It is a radio actively labeled DNA with fluorescent tags which can be used to locate similar / complementary DNA in the genome of any organism
- Useful in diagnosis, DNA probes can hybridize with specific DNA sequence & help diagnose
  - Specific parasite DNA
  - Mutation at specific locations in genomes etc.
- Earlier identification of parasite now became easy as DNA probes have been made for a no. of these parasites
- All made for most of gene disorders / mutation enabling easy diagnosis of these diseases
- DNA probes have been developed for Leishmania, Trypanosoma,Plasmodium, Schistosoma, Wuchereria and other human parasites
- DNA probes are also used in DNA Profiling
- DNA probes are made by PCR
RFLP (Restriction Fragment Length Polymorphism) – has been used in study of genetic variation in man.
- Since different individuals differe in DNA bases every 200 basepairs, it follows that different length of DNA fragments will be produced if cleaved by same restriction endonuclease. it is referred to as RFLP.
- This can be recongnized by the altered mobility of the DNA fragments in the electric field, such as gel electrophoresis, where rate of migration in gel depend on their sizes.
- Smaller fragments travel longer distance b/c of less frictional force & become aranged in the gel in the form of smear – from larger to smaller size.
- RFLPs can be studies in following four ways
  - RFLP for deletion detection
  - Allele linked RFLP
  - Locus Linked RFLP
  - Mutation sepcific RFLP
VNTR (Variable Number Tandem Repeat)
- This is also k/n as hypervariable region in which a core sequence of 10-15 base pairs is repeated.
- Such hypervariable regions are distributed throughout the genome, its no. & distribution varying in different individuals
- Like other genes, VNTRs are also inherited in Mendelian fashion ; hence progeny tend to inherit a particular pattern fron their parents.
- If DNA is cleaved, VNTRs go along with fragments of different sizes in different individuals & when probed with complementary regions, give different pattern.
- Besides being useful in DNA finger printing, VNTRs are also linked to certain allele & presence of specific size of DNA fragment with VNTR is indicative of presence of that allele.
CA Repeats – Certain VNTRs are small, consisting of repeats of two bases – CA, GT etc.
- Human Genome contains 50 thousand to 1 lakh blocks of such repeats.The CA repeats have also been found linked to certain disease locus.
Gene Therapy
- In addition to producing transgenic organism like disease resistant plants, high yielding food crops, oil eating bacteria
- rDNA technology has enormous potential in human diseases & medicine
- Production of Hormones
  - issulin (diabtetes)
  - growth hormone (dwarfism)
  - Factor VIII (Hemophilia)
  - Eurokinase (thrombosis)
  - interferon (infections)
  - Somatostatin (gigantism)
- Production of Synthetic Vacciens
  - vaccine against Hepatitis B,
  - Foot & mouth disease,
  - Feline leukemia
  - virus vaccine

Advanced Molecular Technologies Anthropological Techniques

– Comparative serology, genetic sequences or protein sequences

– As it allow to determine closeness of relationships

– Geographical origin of population & trance migration pattern

– Thus helped in establishing evolutionary tree & hypothesis about evolutionary relatedness of org

– Mitochondrial study – trancing maternal inheritance only

– Also being used to study origin of modern humans

– Polymerase Chain Reaction (PCR)

– molecular hybridisation

– Gene Electrophoresis

– DNA Finger printing

– DNA technology – (Genetic engineering)

– Ex – genetic engineered insulin

– Genetic therapy

– Ex – ADA deficiency

– Gene editing

– Crispr/ cas9

– Genomic imprinting

– Genome sequencing

– Meta – genomes etc

– Also can help in finding cure for disease like HIV, cancer

– Molecular tech also help in Help in cytology

– Ex – erythroblastosis fetalis

– molecular techniques in genetic counseling, genetic screening

9.1: Human Genetics – Method & Application