Advances in Advances in molecular genetics molecular genetics

of poultryof poultry

Presented By :Presented By :

Alok BhartiAlok BhartiMVSc. [ AGB ]MVSc. [ AGB ]

What is molecular What is molecular geneticsgenetics

Is the study of genetic make up of Is the study of genetic make up of individuals at the DNA levelindividuals at the DNA level

The field overlaps with other areas The field overlaps with other areas of biology and of biology and chemistry, chemistry, particularly genetics and particularly genetics and biochemistry.biochemistry.

Beginning of Molecular Beginning of Molecular BiologyBiology

The The Modern of molecular biologyModern of molecular biology begins in the 1930s begins in the 1930s

with the convergence of various, with the convergence of various, previously distinct biological previously distinct biological disciplines: disciplines: biochemistry, genetics, biochemistry, genetics, microbiology, and virologymicrobiology, and virology

With the hope of understanding life at With the hope of understanding life at its most fundamental levelits most fundamental level

Warren WeaverWarren Weaver While molecular While molecular

biology was biology was established in the established in the 1930s, the term 1930s, the term was first coined by was first coined by Warren WeaverWarren Weaver in in 19381938

Early Vision on Molecular Early Vision on Molecular BiologyBiology

Conventional methods Conventional methods

Depends on phenotypic selection. ThisDepends on phenotypic selection. This Narrows the genetic base of a population. Narrows the genetic base of a population.

i.e. i.e. Influenced by environmental selective Influenced by environmental selective pressurepressure

Only be applied to traits that are easily Only be applied to traits that are easily measuredmeasured

Takes long period of timeTakes long period of time High costsHigh costs

Limited in Limited in sensitivitysensitivity specificityspecificity

Advantages of molecular Advantages of molecular geneticsgenetics

Not affects by environmental effects. i.e. Not affects by environmental effects. i.e. hh22=1.=1.

Can be available at an early age (in Can be available at an early age (in principle at the embryo stage)principle at the embryo stage) Allowing early selection Allowing early selection Generation interval reductionGeneration interval reduction

Especially beneficial for sex-limited traits, Especially beneficial for sex-limited traits, traits that are difficult to record, or traits traits that are difficult to record, or traits that require slaughter of the animal that require slaughter of the animal (carcass traits)(carcass traits)

Scope of molecular Scope of molecular geneticsgenetics

Molecular analysis of genetic diversityMolecular analysis of genetic diversity Animal identification and traceabilityAnimal identification and traceability Reproductive enhancement Reproductive enhancement Transgenic livestockTransgenic livestock Germ line manipulationGerm line manipulation Gene based trait selectionGene based trait selection Poultry health: diagnosis , protection Poultry health: diagnosis , protection

and treatment and treatment

Molecular genetics toolsMolecular genetics tools

Molecular Genetics

Cloning Technology

Cloning Technology

DNA Sequencin

g Technology

DNA Sequencin

g Technology

Transgenesis

Technology

Transgenesis

Technology

PCR Technologie

s

PCR Technologie

sMarker Assisted Selection

Marker Assisted Selection

Introduction : Molecular Markers

Also known as a genetic marker.

It usually applies to an allele, DNA marker, or cytogenetic marker

 used as an experimental probe to keep track of an individual, a tissue, a cell, a nucleus, a chromosome, or a gene. 

Molecular markers are chosen because they are

Relatively cheap Amenable to High throughput

protocols Distributed over most of

genome Not dependent on high DNA

sample availability

Why do we care about variations?

underlie phenotypic differences

cause inherited diseases

allow tracking human/animal history (ancient and modern)

Molecular Marker Molecular Marker TechniquesTechniques

1)1) Restriction Fragment Length Restriction Fragment Length Polymorphism (RFLP)Polymorphism (RFLP)

The technique centers around the digestion of The technique centers around the digestion of genomic DNA digested with restriction enzymes genomic DNA digested with restriction enzymes [Forming recognition sites][Forming recognition sites]

These recognition sites are not associated with any These recognition sites are not associated with any type of gene and are distributed randomly type of gene and are distributed randomly throughout the genome. throughout the genome.

When genomic DNA is digested with one of these When genomic DNA is digested with one of these restriction enzymes, (of which there are thousands, restriction enzymes, (of which there are thousands, each cutting at a specific sequence), a series of each cutting at a specific sequence), a series of fragment are produced of varying length.fragment are produced of varying length.

These fragments are separated using PAGE These fragments are separated using PAGE and yield a characteristic patternand yield a characteristic pattern

Variations in the characteristic pattern of a Variations in the characteristic pattern of a RFLP digest can be caused by RFLP digest can be caused by base pair deletions, base pair deletions, mutations, mutations, inversions, inversions, translocations and transpositions translocations and transpositions

which result in the loss or gain of a which result in the loss or gain of a recognition site resulting in a fragment of recognition site resulting in a fragment of different length and polymorphism.different length and polymorphism.

RFLPRFLP

6-cutter GAATTC6-cutter GAATTC 4-cutter TCGA4-cutter TCGA CTTAAGCTTAAG AGCTAGCT

Enzymes cut DNA at specific sequencesRestriction sites are often palindromes:

Advantages:Advantages:

variants are co-dominant; variants are co-dominant; measures variation at the level of DNA sequence, measures variation at the level of DNA sequence,

not protein sequence.not protein sequence.

Disadvantages:Disadvantages:

labor intensive; labor intensive; requires relatively large amounts of DNArequires relatively large amounts of DNA

Using RFLP polymorphismUsing RFLP polymorphism

PCR Based Molecular MarkersPCR Based Molecular Markers

2. Randomly amplified polymorphic DNA 2. Randomly amplified polymorphic DNA Markers (RAPD)Markers (RAPD)

RAPD was the first PCR based molecular marker RAPD was the first PCR based molecular marker technique developed and it is by far the simplest. technique developed and it is by far the simplest.

Short PCR primers (approximately 10 bases) are Short PCR primers (approximately 10 bases) are randomly and arbitrarily selected to amplify random randomly and arbitrarily selected to amplify random DNA segments throughout the genome. DNA segments throughout the genome.

PCR Based Molecular MarkersPCR Based Molecular Markers

The resulting amplification product is generated The resulting amplification product is generated at the region flanking a part of the 10 bp priming at the region flanking a part of the 10 bp priming sites in the appropriate orientation. sites in the appropriate orientation.

RAPD often shows a dominant relationship due to RAPD often shows a dominant relationship due to primer being unable to bind on recessive alleles. primer being unable to bind on recessive alleles.

RAPD products are usually visualized on agarose RAPD products are usually visualized on agarose gels stained with ethidium bromide.gels stained with ethidium bromide.

RAPD: Randomly amplified polymorphic RAPD: Randomly amplified polymorphic DNADNA

Size sorted

RAPDsRAPDs

Advantages:Advantages:

fast, fast, relatively inexpensive, relatively inexpensive, highly variable.highly variable.

Disadvantages:Disadvantages:

markers are markers are dominant. dominant. Presence of a band could mean the individual is Presence of a band could mean the individual is

either heterozygous or homozygous for the either heterozygous or homozygous for the sequence--can’t tell which. sequence--can’t tell which.

Data analysis more complicated.Data analysis more complicated.

B

RAPD AnalysisRAPD Analysis

3. Simple Sequence Repeats 3. Simple Sequence Repeats (SSR)/Microsatellites(SSR)/Microsatellites

Simple sequence repeats are present in the Simple sequence repeats are present in the genomes of all eukaryotes and consists of several genomes of all eukaryotes and consists of several to over a hundred repeats of a 1-4 nucleotide to over a hundred repeats of a 1-4 nucleotide motif.motif.

4. Amplified Fragment Length 4. Amplified Fragment Length Polymorphism (AFLP)Polymorphism (AFLP)

AFLP is the latest form of marker assisted AFLP is the latest form of marker assisted selection and is a highly sensitive method selection and is a highly sensitive method based on the combined concepts of RFLP based on the combined concepts of RFLP and RAPDand RAPD

This technique is applicable to all species This technique is applicable to all species giving very reproducible resultsgiving very reproducible results

The basis of AFLP is the PCR The basis of AFLP is the PCR amplification of restriction enzyme amplification of restriction enzyme fragments of genomic DNAfragments of genomic DNA

AFLPsAFLPs

AFLPsAFLPs

Advantages:Advantages:

fast, fast, relatively inexpensive, relatively inexpensive, highly variable.highly variable.

DisadvantagesDisadvantages: :

markers are markers are dominant. dominant. Presence of a band could mean the individual is Presence of a band could mean the individual is

either heterozygous or homozygous for the either heterozygous or homozygous for the sequence--can’t tell whichsequence--can’t tell which..

VNTR: variable number tandem VNTR: variable number tandem repeatsrepeats

Non-coding regionsNon-coding regions

Several to many copies of the same Several to many copies of the same sequencesequence

Large amount of variation among Large amount of variation among individuals in the individuals in the number of copiesnumber of copies

VNTRMini satellite

MicrosatellitesMicrosatellites

Design primers to flanking regionsDesign primers to flanking regions

MicrosatellitesMicrosatellites

Used for within-Used for within-population studies; population studies;

not as much for not as much for between-population between-population studies b/c they studies b/c they evolve too fastevolve too fast

Paternity analysis and Paternity analysis and other studies of other studies of kinshipkinship

MicrosatellitesMicrosatellites

Advantages: Advantages:

highly variable, highly variable, fast evolving, fast evolving,

Disadvantages:Disadvantages:

Relatively expensive and time consuming to Relatively expensive and time consuming to developdevelop

What is a SNP?

SNP Key ConceptsSNP Key Concepts

Definition: More than one alternative bases occur at an appreciable frequency

Availability: Over 10 million SNPs have been identified in human genome (dbSNP Build 125)

Function: Most SNPs are neutral, and less than 1% is present in protein-coding regions

SNPSNP The most common genetic polymorphismThe most common genetic polymorphism Distribute throughout genome with high Distribute throughout genome with high

densitydensity More stable and easy to assay More stable and easy to assay Major cause of genetic diversity among Major cause of genetic diversity among

different (normal) individuals, e.g. drug different (normal) individuals, e.g. drug response, disease susceptibility.response, disease susceptibility.

Facilitates large scale genetic association Facilitates large scale genetic association studies as genetic markersstudies as genetic markers..

SNP Types

Application of MASApplication of MAS

Parentage determinationParentage determination Estimation of genetic distanceEstimation of genetic distance Sex determinationSex determination Identification of carrier stageIdentification of carrier stage Gene mappingGene mapping

Polymerase Chain ReactionPolymerase Chain Reaction

Polymerase chain reactionPolymerase chain reaction

Polymerase chain reactionPolymerase chain reaction was invented was invented by Kary Mullis and his colleagues in the by Kary Mullis and his colleagues in the 1983. 1983. Nobel prize 1993Nobel prize 1993

It has become the most widely used It has become the most widely used nucleic acid amplification technologynucleic acid amplification technology

PCRPCR is a technique for is a technique for in vitroin vitro amplification of specific DNA sequences amplification of specific DNA sequences

. .

TemperatureTemperature mediated mediated DNA polymeraseDNA polymerase enzyme by simultaneous primer extension enzyme by simultaneous primer extension of complementary strands of DNAof complementary strands of DNA

PCRPCR is a is a test tubetest tube system for DNA system for DNA replication that allows a replication that allows a "target""target" DNA DNA sequence to be selectively amplified, sequence to be selectively amplified, several million-fold in just a few hoursseveral million-fold in just a few hours

Standard Polymerase Chain ReactionStandard Polymerase Chain Reaction

PROCEDURE

DENATURATION

ANNEALINGDNA SYNTHESIS

PCR Reaction ComponentsPCR Reaction Components

Template:Template: previously isolated and purified. previously isolated and purified. Two primers:Two primers: to flank the target sequence.to flank the target sequence. Four deoxynucleosides triphosphate Four deoxynucleosides triphosphate

(dNTPs):(dNTPs): to provide energy and nucleosides to provide energy and nucleosides for the synthesis of DNA.for the synthesis of DNA.

Buffer systemBuffer system containing magnesium.containing magnesium. DNA polymeraseDNA polymerase

PCR Reaction ComponentsPCR Reaction Components

TemplateTemplate The recommended amount of template for The recommended amount of template for standard PCR isstandard PCR is::

The maximum amount AnimalThe maximum amount Animal genomic DNA should be up to genomic DNA should be up to 500 ng.500 ng.

1-10 ng bacterial DNA.1-10 ng bacterial DNA. 0.1-1 ng plasmid DNA.0.1-1 ng plasmid DNA.

Primers:Primers: Primer concentration between Primer concentration between 0.1 and 0.6 0.1 and 0.6 M M are are generally generally optimal.optimal.

Higher primer concentrations may promote mispriming and Higher primer concentrations may promote mispriming and accumulation of non specific productaccumulation of non specific product

Lower primer concentrations may be exhausted before the Lower primer concentrations may be exhausted before the reaction is reaction is completed, resulting in lower yield of desired completed, resulting in lower yield of desired productproduct

Deoxynucleosides Triphosphate (dNTPs) Deoxynucleosides Triphosphate (dNTPs) ConcentrationConcentration

Balanced solution of all four dNTPs minimize Balanced solution of all four dNTPs minimize polymerase error ratepolymerase error rate

Buffer systemBuffer system containing magnesium:containing magnesium:Providing a suitable chemical Providing a suitable chemical

environment for optimum activity and environment for optimum activity and stability of the DNA polymerase. stability of the DNA polymerase. Generally, Generally, the Ph of the reaction buffer is (Ph 8.3 – 9.0) the Ph of the reaction buffer is (Ph 8.3 – 9.0) will give the optimal results.will give the optimal results.

Mg+ ConcentrationMg+ Concentration The optimal MgClThe optimal MgCl22 concentration may concentration may

vary from approximately 1mM-5mM, 1.5 mM vary from approximately 1mM-5mM, 1.5 mM is optimal in most cases.is optimal in most cases.

DNA Polymerase: DNA Polymerase: The recommended amount is 0.5 – 2.5 The recommended amount is 0.5 – 2.5

units/50 units/50 l l reaction. Too little will limit the reaction. Too little will limit the amount of product, while too much can amount of product, while too much can produce unwanted non specific products and produce unwanted non specific products and decreased specificity.decreased specificity.

DNA PolymeraseDNA Polymerase

The most widely characterized polymerase is The most widely characterized polymerase is that fromthat from Thermus aquaticusThermus aquaticus (Taq DNA (Taq DNA Polymerase),Polymerase), which is a which is a thermophilic thermophilic bacterium bacterium lives in hot springs and capable lives in hot springs and capable of growing at 70 -75 C of growing at 70 -75 C . .

The purified proteinThe purified protein (Taq enzyme) consist of (Taq enzyme) consist of a single polypeptide chaina single polypeptide chain has a molecular has a molecular weight of 95 Kd, and has an optimum weight of 95 Kd, and has an optimum polymerization temperature of 70 – 80 C polymerization temperature of 70 – 80 C ..

Initial Denaturation:Initial Denaturation: This step consists of heating the reaction to a temperature This step consists of heating the reaction to a temperature

of 94-95°C which is held for 1-9 minutesof 94-95°C which is held for 1-9 minutesIt is enough to completely denature complex genomic DNAIt is enough to completely denature complex genomic DNA

Each cycle includes three successive steps:Each cycle includes three successive steps:Each cycle takes as little as few minutes and it usually takes Each cycle takes as little as few minutes and it usually takes fewer than 20 cycles to produce as much amplified DNA as fewer than 20 cycles to produce as much amplified DNA as one needsone needs

Denaturation:Denaturation: One to several minutes at 94-96 COne to several minutes at 94-96 C, during , during which the DNA is denatured into single strandswhich the DNA is denatured into single strands

Annealing:Annealing: One to several minutes at 50-65 C One to several minutes at 50-65 C , during which , during which the primers hybridize or "anneal" (by way of hydrogen the primers hybridize or "anneal" (by way of hydrogen bonds) to their complementary sequences on either side of bonds) to their complementary sequences on either side of the target sequencethe target sequence

Extention:Extention: For fragments up to 3 kb, primer extension is For fragments up to 3 kb, primer extension is normally carried out at 72 C normally carried out at 72 C Polymerase binds and extends a complementary DNA strand Polymerase binds and extends a complementary DNA strand from each primer and add approximately 60 bases per from each primer and add approximately 60 bases per second at 72C second at 72C . .

Thermal Cycling Profile for Standard PCR

DenaturatioDenaturationn

AnnealinAnnealingg

ExtentioExtentionn

Number of Cycles:Number of Cycles:

The number of cycles required for optimum amplification The number of cycles required for optimum amplification varies depending on the amount of the starting material. varies depending on the amount of the starting material.

In optimal reaction, less than 10 template molecules can In optimal reaction, less than 10 template molecules can be amplified in less than 40 cycles to a product that is be amplified in less than 40 cycles to a product that is easily detectable on a gel stained with ethidium bromide. easily detectable on a gel stained with ethidium bromide.

Most PCR should , therefore, include only 25 – 35 cycles. Most PCR should , therefore, include only 25 – 35 cycles.

As cycle increases, nonspecific products can accumulate.As cycle increases, nonspecific products can accumulate. After 20- 40 cycles of heating and cooling build up over a After 20- 40 cycles of heating and cooling build up over a

million copies of original DNA molecules.million copies of original DNA molecules.

PCR PhasesPCR PhasesThree phases:Three phases:

Exponential:Exponential: Exact doubling of product is accumulating at Exact doubling of product is accumulating at every cycle (assuming 100% reaction efficiency). The every cycle (assuming 100% reaction efficiency). The reaction is very specific and precisereaction is very specific and precise

Linear:Linear: The reaction components are being consumed, the The reaction components are being consumed, the reaction is slowing, and products are starting to degradereaction is slowing, and products are starting to degrade

Plateau:Plateau: The reaction has stopped, no more products are The reaction has stopped, no more products are being made and if left long enough, the PCR products will being made and if left long enough, the PCR products will begin to degradebegin to degrade

Advantages of PCR:Advantages of PCR:

Useful non- invasive procedure. Useful non- invasive procedure. Simplicity of the procedure.Simplicity of the procedure. Sensitivity of the PCR.Sensitivity of the PCR.

Disadvantages of PCR:Disadvantages of PCR:

False positive results (cross contamination)False positive results (cross contamination) False negative results (e.g. rare of circulating False negative results (e.g. rare of circulating

fetal cells)fetal cells)

Variants of PCRVariants of PCR

Reverse Reverse transcriptase-PCRtranscriptase-PCR

Nested-PCR.Nested-PCR.

Hot-start PCRHot-start PCR

Quantitative PCRQuantitative PCR

Multiplex-PCRMultiplex-PCR

Mutagenesis by PCR

Allele specific PCR

Inverse PCR

Asymmetric PCR

In Situ PCR

Reverse Transcriptase-PCRReverse Transcriptase-PCR

RT-PCR,RT-PCR, one of the most sensitive methods for one of the most sensitive methods for the detection and analysis of rare mRNA the detection and analysis of rare mRNA transcripts or other RNA present in low transcripts or other RNA present in low abundance.abundance.

RNARNA cannot serve as a template for PCR, so it cannot serve as a template for PCR, so it must be first transcribed into cDNA with must be first transcribed into cDNA with reverse transcriptase and the cDNA copy is reverse transcriptase and the cDNA copy is then amplified.then amplified.

The technique is usually initiated by mixing The technique is usually initiated by mixing short (12-18 base) polymers of thymidine short (12-18 base) polymers of thymidine (oligo (oligo dT)dT) with messenger RNA such that they anneal with messenger RNA such that they anneal to the RNA's polyadenylate tail. Reverse to the RNA's polyadenylate tail. Reverse transcriptase is then added and uses the oligo transcriptase is then added and uses the oligo dT as a primer to synthesize so-called dT as a primer to synthesize so-called first-first-strand cDNAstrand cDNA..

Nested PCRNested PCR

Nested PCRNested PCR is a variation of is a variation of the polymerase chain the polymerase chain reaction (PCR), in that two reaction (PCR), in that two pairs (instead of one pair) of pairs (instead of one pair) of PCR primers are used to PCR primers are used to amplify a fragment.amplify a fragment.

The first pair of PCR The first pair of PCR primers amplify a fragment primers amplify a fragment similar to a standard PCR. similar to a standard PCR. However, a second pair of However, a second pair of primers called nested primers called nested primers bind inside the first primers bind inside the first PCR product fragment to PCR product fragment to allow amplification of a allow amplification of a second PCR product which second PCR product which is shorter than the first one.is shorter than the first one.

Nested PCR is a very specific Nested PCR is a very specific PCR amplificationPCR amplification.. http://www.pcrstation.com/images/nested-pcr.gif

Hot Start PCRHot Start PCR Hot Start PCRHot Start PCR significantly significantly

improve specificity, improve specificity, sensitivity and yield of PCRsensitivity and yield of PCR

Some components essential Some components essential for polymerase activity is for polymerase activity is separated from the reaction separated from the reaction mixture until the mixture until the temperature in the tubes has temperature in the tubes has exceeded the optimal primer exceeded the optimal primer annealing temperature annealing temperature usually 55-65 C ˚usually 55-65 C ˚

Specialized enzyme systems Specialized enzyme systems have been developed that have been developed that inhibit the polymerase's inhibit the polymerase's activity at ambient activity at ambient temperature, either by the temperature, either by the binding of an binding of an antibody antibody or by or by the presence of covalently the presence of covalently bound inhibitors that only bound inhibitors that only dissociate after a high-dissociate after a high-temperature activation steptemperature activation step..

Quantitative PCRQuantitative PCR

The determination or quantitation of the number of copies The determination or quantitation of the number of copies of a given gene achieves accurate estimation of DNA and of a given gene achieves accurate estimation of DNA and RNA targets.RNA targets.

Real Time PCRReal Time PCR Traditional PCR has advanced from detection at the end-Traditional PCR has advanced from detection at the end-

point of the reaction. to detection while the reaction is point of the reaction. to detection while the reaction is occurringoccurring Real-Time PCR Real-Time PCR is used.is used.

Real-time PCR uses a fluorescent reporter signal to Real-time PCR uses a fluorescent reporter signal to measure the amount ofmeasure the amount of ampliconamplicon as it is generatedas it is generated .. This This kinetic PCRkinetic PCR allows for data collection after each cycle of allows for data collection after each cycle of PCR instead of only at the end of the 20 to 40 cycles.PCR instead of only at the end of the 20 to 40 cycles.

The real time system reduces the time required for The real time system reduces the time required for PCR amplification and analysis from hours to PCR amplification and analysis from hours to minutes.minutes.

Monitor amplification online and in real-time.Monitor amplification online and in real-time.

Quickly and accurately quantify results. Quickly and accurately quantify results.

Analyze melting characteristics of PCR product.Analyze melting characteristics of PCR product.

The recent development of real time PCR clearly The recent development of real time PCR clearly demonstrates demonstrates many advantages over other existing method with: many advantages over other existing method with: high accuracy, high accuracy, wide dynamic range , specificity , sensitivity , reduced wide dynamic range , specificity , sensitivity , reduced carry over contamination and rapid accurate and carry over contamination and rapid accurate and simultaneous quantification of multiple samples.simultaneous quantification of multiple samples.

Multiplex PCRMultiplex PCR , made it possible to compare two or , made it possible to compare two or more complex genomes, for instance to detect more complex genomes, for instance to detect chromosomal imbalances.chromosomal imbalances.

Combining in situ hybridization with PCR made Combining in situ hybridization with PCR made possible the localization of single nucleic acid possible the localization of single nucleic acid sequences on one chromosome within an eukaryotic sequences on one chromosome within an eukaryotic organism.organism.

Amplification ofAmplification of archivalarchival and and forensicforensic material.material.

• Identify testing for transplantation, Identify testing for transplantation, HLAHLA Typing.Typing.

• PCR is used in research laboratories in DNA cloning PCR is used in research laboratories in DNA cloning procedures, Southern blotting, DNA sequencing, procedures, Southern blotting, DNA sequencing, recombinant DNA technology.recombinant DNA technology.

Multiplex PCR

Polymerase Chain ReactionPolymerase Chain Reaction

The polymerase chain reaction The polymerase chain reaction (PCR)(PCR) is a technique is a technique widely used inwidely used in::

Molecular biology, Molecular biology, Microbiology, Microbiology,

Genetics, Genetics, Diagnostics clinical laboratories, Diagnostics clinical laboratories,

Forensic science, Forensic science, Environmental science, Environmental science,

Hereditary studies, Hereditary studies, Paternity testing, and Paternity testing, and

Many other Many other applications……………………………………… applications………………………………………

DNA SEQUENCINGDNA SEQUENCING

How DNA Sequence Is Determined?

Polyacrylamide Gel Electrophoresis

ATC32PAT32PA32P

T A G C

T

A

CG

ATCG32P

ATCGA32P

ATCGAT32P

ATCGATC32P

ATCGATCG32PATCGATCGA32P

ATCGATCGAT32P

DNA fragments having a difference of one nucleotide can be separated on gel electrophoresis

But these bands can’t tell usthe identity of the terminal nucleotides

If those band with the sameterminal nucleotide can begrouped, then it is possible to read the whole sequence

Juang RH (2004) BCbasics

How to Obtain DNA Fragments

AT

ATCGAT

Specific Reaction to G

A

Terminated

Biosynthetic method

Chemical method

Template

or

Non-radioactive(invisible)

Producing various fragments

Phosphodiesterbond

P R P R P R P R P R P R

OH

5’

3’

1

3’

5’

A

1

2

3

4

5

6

APO4

2-

H3’

5’

2

Hdideoxynuceotide

TerminatedTerminatedddNTP

Sanger’s M

ethod: How

Term

inated

Normal

Linking

Can not reactJuang RH (2004) BCbasics

Application of DNA sequencing Application of DNA sequencing technologytechnology

Determination of exact order of Determination of exact order of bases i.e. deciphering the genetic bases i.e. deciphering the genetic blueprint of humans, plants, animals blueprint of humans, plants, animals and micro-organisms.and micro-organisms.

Conservation of gene sequencesConservation of gene sequences

GENE CLONING

Gene Transferred by Plasmid

Plasmid gets out and into the host cell

Resistant Strain

New Resistance Strain

Non-resistant Strain

Plasmid

EnzymeHydrolyzingAntibiotics

Drug Resistant Gene

mRNA

Juang RH (2004) BCbasics

Target Genes Carried by Plasmid

1 plasmid1 cellRecombinant

PlasmidTransformation

Target GeneRecombination

Restriction

Enzyme

Restriction

Enzyme

Ch

rom

oso

mal

DN

ATarget Genes

DNA Recombination

TransformationHost Cells

Amplification and Screening of Target Gene

1

1 cell line, 1 colonyX100

X1,000

PlasmidDuplicationBacteria

Duplication

Plating

Pick the colonycontaining target gene

=100,000

Species clonedSpecies cloned CattleCattle: Alpha and Beta (males, 2001) and (2005) Brazil: Alpha and Beta (males, 2001) and (2005) Brazil CatCat: CopyCat "CC" (female, late 2001), Little Nicky, : CopyCat "CC" (female, late 2001), Little Nicky,

2004, 2004, was the first cat cloned for commercial was the first cat cloned for commercial reasonsreasons

MuleMule: Idaho Gem, a john mule born 4 May 2003, was : Idaho Gem, a john mule born 4 May 2003, was the first the first horse-family clone.horse-family clone.

HorseHorse: Prometea, a Haflinger female born 28 May : Prometea, a Haflinger female born 28 May Water BuffaloWater Buffalo: Samrupa was the first cloned water : Samrupa was the first cloned water

buffalo. It buffalo. It was born on February 6, 2009, at India's was born on February 6, 2009, at India's Karnal, NDRI Karnal, NDRI but died five days later due to lung but died five days later due to lung infection.infection.

CamelCamel: (2009) Injaz, is the first cloned camel2003, was : (2009) Injaz, is the first cloned camel2003, was the first horse clone.the first horse clone.

Application of gene Application of gene cloningcloning

Identifying, localizing and Identifying, localizing and characterizing genescharacterizing genes

Creating animal models for studying Creating animal models for studying genetic diseasesgenetic diseases AgingAging CancerCancer therapeutic effects of drugstherapeutic effects of drugs

Providing animal researchers a tool Providing animal researchers a tool for saving endangered speciesfor saving endangered species

Transgenesis

Process of introducing foreign or exogenous DNA into an animal’s genome

Transgene : DNA introduced

•Mice

•Cows

•Fish

•Birds

•Sheep

•Goats

Why Transgenesis?Why Transgenesis?•Improve genetic Features of domesticated Animals

•Provide animal models for study of human diseases

•Pharmingusing farm animals for production of human

pharmaceuticals-mammary glands

•Study the genes regulation, development of animals

How to Get the Transgene Inserted •Retroviral Vectors

•Microinjection•Embryonic stem cells

MicroinjectionMicroinjection

A.Remove eggs

B.B. Fertilize in vitro

C. DNA is microinjected into male pronucleus (prior to nuclear fusion)100-1000 copies of gene

D. Implant eggs into surrogate

Fig 22.13 The production of transgenic Fig 22.13 The production of transgenic animals by microinjection of DNA into animals by microinjection of DNA into

fertilized eggs.fertilized eggs.© 2003 John Wiley and Sons Publishers

Retroviral VectorsRetroviral Vectors

Infect early stage embryo with replication-defective retrovirus

Limitations only small DNA inserts no regulatory sequences safety

Engineered Embryonic Engineered Embryonic Stem CellsStem Cells

• Remove pluripotent ES cells from blastocyst

• Transfect ES

• Selection

• Microinject back into blastocyst

• Implant

Mice make Human Mice make Human AntibodiesAntibodies

YACs contained many of these heavy and light chain segments

Knock out Mouse Segments, replace with Human segment genes

Fully human antibody made

Transgenic CattleTransgenic Cattle

Applications•Increasing casein content of milk increase cheese production•Lactose free milk (transgene lactase)•Resistance to bacterial infections•In vivo immunization

•transgene is specific Heavy and Light chain genes which•create An production against a specific antigen

Why Express rProtein in Why Express rProtein in MilkMilk

Easy to purify - few other proteins in milkDoesn’t harm transgenic animal- no change to

physiology

rProtein is authentically modified post-translationally

Large quantities

Renewable source

Sheep and PigsSheep and Pigs

PIGSPST porcine somatotropin (growth hormone)

adverse effects- kidney, stomach, heart, sterility

human Hemoglobin to replace whole blood transfusions

SHEEPIncrease wool production

keratin promotergrowth factor

Organ TransplantOrgan Transplant

Pr

Problem: Rejection

Antibodies from Host bind to Donor OrganElicits Inflammatory ResponseTransplanted Organ Lost

Solution:Transgene in Donor for Complement-Inhibiting Protein

111

Birds and FishBirds and Fish

Birdstraditional methods can not be used

because of avian embryogenesis differences

no ES cells foundALV resistant chickens

transgene - defective ALV genomemakes viral RNA and proteinbut blocks assembly of viral

particlesFish

aquaculturetransgene - growth hormone

Application of Application of TransgenesisTransgenesis

Production of bio-molecules in an Production of bio-molecules in an animal which it normally does not animal which it normally does not producesproduces

Promoting productivityPromoting productivity Reducing costs (disease resistance)Reducing costs (disease resistance)

Molecular genetics - Molecular genetics - POULTRY POULTRY

Chickens have 78 chromosomesChickens have 78 chromosomes The chicken genome comprises 39 The chicken genome comprises 39

pairs of chromosomespairs of chromosomes 8 pairs of macro chromosomes8 pairs of macro chromosomes 1 pair of sex chromosome1 pair of sex chromosome 30 pairs of micro chromosome30 pairs of micro chromosome

Molecular genetics - Molecular genetics - POULTRY POULTRY

The size of the chicken genome is estimated The size of the chicken genome is estimated to be 1.2× 10to be 1.2× 1099 base pairs which is App. 4000 base pairs which is App. 4000 cM in length( 1cM =300kb of DNA in chicken)cM in length( 1cM =300kb of DNA in chicken)

Chicken have played a more significant role Chicken have played a more significant role in molecular genetics than in most areas of in molecular genetics than in most areas of biologybiology

Mendel discovered the working of single Mendel discovered the working of single genes and chicken were the species in which genes and chicken were the species in which this basic concept was applied, e.g. in the this basic concept was applied, e.g. in the selection methods for plumage colour.selection methods for plumage colour.

Genes of interestGenes of interest

With the possibility of producing With the possibility of producing genetically manipulated transgenic genetically manipulated transgenic chickens, it has become important to chickens, it has become important to identify the genes controlling identify the genes controlling economically important traits:economically important traits:

MUSCLE GROWTHMUSCLE GROWTH PRODUCTIONPRODUCTION REPRODUCTIONREPRODUCTION DISEASE RESISTANTDISEASE RESISTANT

Gene structure Gene structure OVALBUMINOVALBUMIN

Major protein of egg whiteMajor protein of egg white

11stst to be discovered to be discovered Exons (intervening sequences) were locatedExons (intervening sequences) were located 5’ end of introns has G-T dinucleotide5’ end of introns has G-T dinucleotide 3’ end of introns has A-G dinucleotide3’ end of introns has A-G dinucleotide polymorphismfor restriction sites within polymorphismfor restriction sites within

introns was found for both an ECoRI site and introns was found for both an ECoRI site and Hae Hae ⅢⅢsitesite

All this holds good for other eukaryotic genes also.All this holds good for other eukaryotic genes also.

Gene structure Gene structure CONALBUMINCONALBUMIN

Major iron binding protein. So, acts as Major iron binding protein. So, acts as antimicrobial agent.antimicrobial agent.

≈ ≈ 10% of the protein10% of the protein Purified by polysome immuno-Purified by polysome immuno-

precipitation and column precipitation and column chromatography from chicken oviduct & chromatography from chicken oviduct & cDNA synthesizedcDNA synthesized

cDNA was used to demonstrate that the cDNA was used to demonstrate that the mRNA in linear and oviduct are identical mRNA in linear and oviduct are identical and coded for by the same geneand coded for by the same gene

Is a protease inhibitor Is a protease inhibitor ≈≈10% of egg white protein10% of egg white protein ** transcription initiates at 2 sites, ** transcription initiates at 2 sites,

one related to a typical TATA box one related to a typical TATA box around -30 base pairs and another around -30 base pairs and another related to a AT rich region 85 related to a AT rich region 85 basepairs upstream, the former basepairs upstream, the former being more efficient in vivo.being more efficient in vivo.

Gene structure Gene structure OVOMUCOIDOVOMUCOID

≈ ≈ 2% of egg white proteins2% of egg white proteins 11stst enzyme whose tertiary structure enzyme whose tertiary structure

was determinedwas determined ** there are multiple mRNA for ** there are multiple mRNA for

chicken lysosome differing at the 5’ chicken lysosome differing at the 5’ end and extending to either 29, 31, end and extending to either 29, 31, or 53 nucleotides from the common or 53 nucleotides from the common initiation codoninitiation codon

Gene structure Gene structure LYSOSOMELYSOSOME

The full genome sequence of The full genome sequence of the chickenthe chicken

2004 -the first draft of the chicken 2004 -the first draft of the chicken genome assembly genome assembly

Large numbers of chicken full-length Large numbers of chicken full-length cDNAs are already beingcDNAs are already being  sequencedsequenced

It has been predicted that the chicken has It has been predicted that the chicken has 35,00035,000  genes in total ~ 50% of these have genes in total ~ 50% of these have a known homologue in another speciesa known homologue in another species

Some AdvancesSome Advances Molecular kits are being generated to study Molecular kits are being generated to study

metabolicfunctions and immune responses metabolicfunctions and immune responses (MIN (MIN et et al.al. 2003 ; NEIMAN  2003 ; NEIMAN etet  al.al. 2003 ) 2003 )

Analyze global gene expression in target Analyze global gene expression in target tissuestissues  of chickens of chickens (COGBURN (COGBURN et al.et al. 2003 ) 2003 )

There are also projects to target gene function There are also projects to target gene function by disrupting and gaining functionsby disrupting and gaining functions  with the use with the use of RNAi methods of RNAi methods  (HUDSON (HUDSON et al.et al. 2002 ; PEKARIK 2002 ; PEKARIK  et al.et al. 2003 ) 2003 )

Single nucleotide polymorphisms within chicken Single nucleotide polymorphisms within chicken genes are beinggenes are being  exploited for the generation of exploited for the generation of candidate genes for quantitativecandidate genes for quantitative  traits traits (EMARA and (EMARA and KIM 2003 ).KIM 2003 ).

Some AdvancesSome Advances There is consequentlyThere is consequently  extensive research into >200 extensive research into >200

chicken quantitative trait locichicken quantitative trait loci  encoding for disease encoding for disease susceptibility, immunology, leanness, susceptibility, immunology, leanness, eggegg  production, etc. (production, etc. (LIU LIU et al.et al. 2001 ; MARIANI  2001 ; MARIANI et al.et al. 2001) 2001)

Chicken DT40 cell lines are avian-leukosis-virus-Chicken DT40 cell lines are avian-leukosis-virus-induced B cellinduced B cell  lines that exhibit a high ratio of lines that exhibit a high ratio of targeted to random integrationtargeted to random integration  of transfected DNA of transfected DNA constructs at homologous loci constructs at homologous loci (DHAR (DHAR et al.et al.  2001 ). 2001 ). 

A feature of DT40A feature of DT40  cell lines, however, is that they cell lines, however, is that they have a high degree of chromosomalhave a high degree of chromosomal  rearrangements rearrangements that, to date, could not be karyotyped. that, to date, could not be karyotyped. (WINDING and (WINDING and BERCHTOLD 2001 )BERCHTOLD 2001 )

Some AdvancesSome Advances

Some 15,000 genes are expressed in Some 15,000 genes are expressed in chicken tissues involved in the regulation chicken tissues involved in the regulation and development of immune and development of immune responsesresponses((http://ec.europa.eu/research/agriculture/projects_showchttp://ec.europa.eu/research/agriculture/projects_showcase01_en.htm)ase01_en.htm)

The chicken"s major histocompatibility complex The chicken"s major histocompatibility complex (MHC) haplotype has a profound influence on the (MHC) haplotype has a profound influence on the resistance or susceptibility to certain resistance or susceptibility to certain pathogenspathogens((Research Project: Research Project: USING THE GENOME TO UNDERSTAND USING THE GENOME TO UNDERSTAND IMMUNOGENETICS OF POULTRY)IMMUNOGENETICS OF POULTRY)

Every body fishing for New Every body fishing for New IdeasIdeas

How much we differ from Ape How much we differ from Ape DNA answersDNA answers ??

April 25th celebrates the DNA ModelApril 25th celebrates the DNA Model DNA continues to the Temple of DNA continues to the Temple of

ScienceScience

Madam, are you fiddling with Madam, are you fiddling with GENES ?GENES ?

Be A GENE GeniusBe A GENE GeniusBecome FamousBecome Famous

REFERENCEREFERENCE www.ensembl.orgwww.ensembl.org Bumstead, N. and Palyga, J. (1992) A preliminary linkage map of the Bumstead, N. and Palyga, J. (1992) A preliminary linkage map of the

chicken genome. Genomics 13: 690-697chicken genome. Genomics 13: 690-697.. Groenen, M.A.M., Cheng, H.H., Bumstead, N., Benkel, B.F., Briles, W.E., Groenen, M.A.M., Cheng, H.H., Bumstead, N., Benkel, B.F., Briles, W.E.,

Burke, T. et al. (2000).A consensus linkage map of the chicken genome. Burke, T. et al. (2000).A consensus linkage map of the chicken genome. Genome Research 10: 137-147.Genome Research 10: 137-147.

Molecular genetics in a modern poultry breeding organization Molecular genetics in a modern poultry breeding organization J.E. FULTONJ.E. FULTONaa

BURT, D. W., C. BRULEY, I. C. DUNN, C. T. JONES, and A. BURT, D. W., C. BRULEY, I. C. DUNN, C. T. JONES, and A. RAMAGERAMAGE et al. et al., 1999  The dynamics of chromosome evolution in , 1999  The dynamics of chromosome evolution in birds and mammals. Nature birds and mammals. Nature 402402:411-413.:411-413.

EMARA, M. G. and H. KIM, 2003  Genetic markers and their EMARA, M. G. and H. KIM, 2003  Genetic markers and their application in poultry breeding. Poult. Sci. application in poultry breeding. Poult. Sci. 8282:952-957:952-957

MASABANDA, J., R. FRIEDL, A. SAZANOV, J. M. LAHTI, and H. MASABANDA, J., R. FRIEDL, A. SAZANOV, J. M. LAHTI, and H. LILI et al. et al., 1998  Mapping of five members of the cyclin gene family , 1998  Mapping of five members of the cyclin gene family on chicken chromosomes by FISH. Chromosome Res. on chicken chromosomes by FISH. Chromosome Res. 66:231-233.:231-233.

REFERENCEREFERENCE

SCHMID, M., I. NANDA, M. GUTTENBACH, C. SCHMID, M., I. NANDA, M. GUTTENBACH, C. STEINLEIN, and M. HOEHNSTEINLEIN, and M. HOEHN et al. et al., 2000  First report on , 2000  First report on chicken genes and chromosomes 2000. Cytogenet. Cell chicken genes and chromosomes 2000. Cytogenet. Cell Genet. Genet. 9090:169-218:169-218

TATSUDA, K. and K. FUJINAKA, 2001  Genetic mapping of TATSUDA, K. and K. FUJINAKA, 2001  Genetic mapping of the QTL affecting body weight in chickens using a F2 the QTL affecting body weight in chickens using a F2 family. Br. Poult. Sci. family. Br. Poult. Sci. 4242:333-337.:333-337.

WINDING, P. and M. W. BERCHTOLD, 2001  The chicken WINDING, P. and M. W. BERCHTOLD, 2001  The chicken B cell line DT40: a novel tool for gene disruption B cell line DT40: a novel tool for gene disruption experiments. J. Immunol. Methods experiments. J. Immunol. Methods 249249:1-16:1-16