Homozygosity mapping using lod score method
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HOMOZYGOSITY MAPPING USING LOD SCORE METHOD. BBS- 6. CONTENTS. INTRODUCTION METHODS OF HOMOZYGOSITY MAPPING HOMOZYGOSITY MAPPER GENETIC LINKAGE LOD SCORE METHOD. LET US START WITH THE BASICS!. KEY TERMS. HOMOZYGOSITY. Containing two identical allelic forms Can be homozygous dominant

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Contents
CONTENTS

  • INTRODUCTION

  • METHODS OF HOMOZYGOSITY MAPPING

  • HOMOZYGOSITY MAPPER

  • GENETIC LINKAGE

  • LOD SCORE METHOD




Homozygosity
HOMOZYGOSITY

  • Containing two identical allelic forms

  • Can be homozygous dominant

  • Can be homozygous recessive

  • PEA PLANT


Heterozygous
HETEROZYGOUS

  • BOTH ALLELES OF A GENE ARE DIFFERENT

  • ONE GENE IS DOMINANT

  • ONE GENE IS RECESSIVE


Genetic mapping
GENETIC MAPPING

  • SETTING A LOCATION WITH RESPECT TO A MARKER

  • PLOTTING DNA FRAGMENTS ON CHROMOSOMES

  • HELPFUL IN PREDICTING A DISEASE



Genetic marker
GENETIC MARKER

  • A GENE OR A DNA SEQUENCE FOR A PARTICULAR TRAIT

  • HAS A PARTICULAR LOCATION ON A CHROMOSOME

  • DETECTION HELPFUL IN PREDICTING A DISEASE


Recombination
RECOMBINATION

  • BREAKING AND REJOINING OF DNA MOLECULES

  • EXAMPLE IS CROSSING OVER

  • EXCHANGE OF GENETIC MATERIAL TAKES PLACE

  • RESULTING MOLECULES ARE CALLED RECOMBINANTS


Recombination frequency
RECOMBINATION FREQUENCY

  • TOTAL NUMBER OF RECOMBINANTS/TOTAL NUMBER OF PROGENIES IN A TEST CROSS

  • USED TO DETERMINE THE GENETIC DISTANCE

  • CREATION OF GENETIC MAP

  • CENTIMORGAN


Homozygosity mapping
HOMOZYGOSITY MAPPING

  • METHOD USED TO DETECT THE DISEASE OF THE HOMOZYGOUS CONDITION

  • HELPFUL FOR THE INHERITED DISORDERS



Three main methods
THREE MAIN METHODS

  • SNP MICROARRAYS

  • RFLP

  • MICROSATELLITE MARKERS


1 snp microarrays
1. SNP MICROARRAYS

  • STANDS FOR SINGLE NUCLEOTIDE POLYMORPHISM

  • INVOLVES A SINGLE PCR METHOD FOLLOWED BY GEL ELECTROPHORESIS

  • TETRA-PRIMER ARMS PCR



Applications
APPLICATIONS

  • HIGH DENSITY SNP ARRAYS FOR GENOTYPING

  • MUTATION IDENTIFICATION BY POSITIONAL CLONING


Bardet beidl syndrome
BARDET BEIDL SYNDROME

  • ABBREVIATED AS BBS

  • CHARACTERIZED BY: OBESITY; PIGMENTARY RETINOPATHY; POLYDACTYLY; HYPOGONADISM

  • RENAL AND CARDIAC ABNORMALITES

  • COGNITIVE IMPAIRMENT


2 rflp
2. RFLP

  • STANDS FOR RESTRICTION FRAGMENT LENGTH POLYMORPHISM

  • USED TO FOLLOW THE PATH OF A SPECIFIC GENE

  • VARIATIONS IN THE HOMOLOGOUS DNA SAMPLES


Method
METHOD

  • CUTTING DNA SAMPLES WITH RESTRICTION ENZYMES

  • SEPERATION BY AGAROSE GEL ELECTROPHORESIS

  • DETERMINING THE NUMBER OF FRAGMENTS AND SIZES


Applications1
APPLICATIONS

  • DNA FINGERPRINTING

  • TRACING ANCESTORY

  • STUDYING EVOLUTION AND MIGRATION

  • DETECTION AND DIAGNOSIS

  • GENETIC MAPPING




Homozygosity mapper
HOMOZYGOSITY MAPPER:

  • Web based approach for homozygosity mapping.

  • Stores markers data in its database…users can upload their SNP files there.

  • Data analysis is quick, detects homozygous alleles, and represents graphically.

  • Zooming in and out of a chromosome.

  • Access:

    • Restricted

    • Public

  • Integrated with GeneDistiller engine


Microsatellite markers
Microsatellite Markers:

  • SSTRs

  • VSTMs

  • Acting as markers

  • Di, tri, tetra, penta nucleotides

  • Present on non-coding sequences

  • Amplified by locus specific primers with PCR

  • Example:

  • Presence of AC (n) in birds where n varies from 8 to 50.


Homozygosity mapping using lod score method

  • Uses:

  • Important most tool in mapping genome

  • Serve in biomedical diagnosis as markers for certain disease conditions

  • Primary marker for DNA testing in forensics for high specificity.

  • Markers for parentage analysis

  • address questions concerning degree of relatedness of individuals or groups


Pedigree
Pedigree

  • Lineage or Genealogical study of family lines.

  • Gives list or family tree of ancestors.

  • Used for studies of certain inheritance pattern.


Genetic linkage
Genetic Linkage:

  • Staying together of physically close loci.

  • Offspring acquires more parental combinations.

  • Discovery:

    • An Exception to “Mendel's Law of independent Assortment”

  • Thomas Morgan : Linked genes are physical objects, linked in close proximity


Genetic linkage1
Genetic Linkage

  • Morgan’s Experiments:

    • 1st Cross:

    • F1 Progeny:

      • Heterozygous red eyed males and females

  • 2nd Cross:

  • F2 Progeny:

  • 2,459 red-eyed females

  • 1,011 red-eyed males

  • 782 white-eyed males


Homozygosity mapping using lod score method

  • 129 red-eyed females

  • 132 red-eyed males

  • 88 white-eyed females

  • 86 white-eyed males

  • Conclusions:

  • Eye color is Sex Linked….

  • Physically closer genes do not assort independently


Linkage map
LINKAGE MAP

  • Genetic Map for location determination of genes and genetic markers.

  • Based on markers recombination frequency during cross over.

  • Predicts the relative position, not the physical distance between genes.

  • separated

  • Lesser the distance, more tightly they are bound, more often inherited together.

  • Centi Morgan: unit to calculate linkage distance

    • One centimorgan corresponds to about 1 million base pairs in humans.

    • Two markers on a chromosome are one centimorgan apart if they have a 1% chance of being


Constructing linkage map
Constructing Linkage Map:

  • Based on frequency of genetic markers passing together.


Lod score method
LOD Score Method

  • Developed by Newton E. Morton

  • LOD:Logarithm (base 10) Of Odds

  • A statistical test for linkage analysis in

    • Human

    • Animal

    • Plant populations

  • It checks whether the two loci are:

    • Indeed linked or

    • They occur together by chance

    • Usually done to check linkage of symptoms in syndromes


  • Homozygosity mapping using lod score method

    LOD Score Method

    • The Method:

      • Establish a pedigree

      • Make a number of estimates of recombination frequency

      • Calculate a LOD score for each estimate

      • The estimate with the highest LOD score will be considered the best estimate


    Calculations
    Calculations:

    Where:

    • NR denotes the number of non-recombinant offspring

    • R denotes the number of recombinant offspring

    • Theta is the recombinant fraction, it is equal to R / (NR + R)

    • 0.5 in the denominator means that alleles that are completely unlinked have a 50% chance of recombination


    Lod score result
    LOD score Result

    • LOD score can be either positive or negative

      • Positive LOD score means Linkage present

      • Negative LOD score means No Linkage

    • >3 Evidence for linkage

    • +3 1000 to 1 odds that the linkage did not occur by chance

    • <-2 Evidence to exclude linkage


    Mapping genes with lod score method
    Mapping Genes with LOD Score Method

    • Determines R (Recombination Fraction, fraction of gametes that are recombinant) using data from small families

    • R value varies from 0 – 0.5

      • 0 2 completely linked genes

      • 0.5 2 completely unlinked genes


    Steps involved
    Steps Involved

    • Determine the expected frequencies of F2 phenotypes

    • Determine the likelihood that the family data observed resulted form given R value

    • Determine LOD ratio

    • Add LOD scores from different families to achieve a high LOD score so a most likely R value can be assigned


    Example1
    EXAMPLE

    • We are using two COMPLETELY DOMINANT GENES

    • Heterozygote is indistinguishable from dominant homozygote

    • Two genes are

      • A: with A and a alleles

      • B: with B and b alleles


    Homozygosity mapping using lod score method

    AB

    ab

    P1: AABB X aabb

    Gametes

    F1 AaBb

    Parental Combinations

    Recombinants

    AB

    Ab

    aB

    ab


    Step i calculate the expected frequency of offspring for values of r from 0 0 5
    STEP I: Calculate the expected frequency of offspring for values of R from 0-0.5

    • Determine the frequency of each gamete produced by F1 generation

    • For example if R=0.20, then 20% of the gametes produced will be recombinants which in our example are Ab and aB.

    • As there are 2 types of recombinant gametes, frequency of each type will be 0.10

    • 80% gametes are parental, [AB and ab type] frequency of each of them is 0.40 or 40%


    Homozygosity mapping using lod score method

    Draw values of R from 0-0.5Punnet Square to determine offspring


    Homozygosity mapping using lod score method


    Homozygosity mapping using lod score method
    STEP II: Examine the observed Family Data in light of expected distribution of offspring for each R value

    • Done by determining the likelihood (L)

    • Likelihood:

      • the probability of the observed family

      • determined using the multinomial theorem

        • an extension of the binomial theorem.


    Homozygosity mapping using lod score method

    • First define the terms for the observed family expected distribution of offspring for each R value

      • a = number of A_ B_ offspring

      • b = number of A_ bb offspring

      • c = number of aaB_ offspring

      • d = number of aabb offspring

      • n = total offspring (= a+b+c+d)

    • Define the terms for the expected family proportions

      • p = expected proportion of A_B_ offspring

      • q = expected proportion of A_ bb offspring

      • r = expected proportion of aaB_ offspring

      • s = expected proportion of aabb offspring


    Multinomial theorem
    Multinomial Theorem expected distribution of offspring for each R value

    • Multinomial theorem describing actual family: paqbrcsdmultiplied by a coefficientn! /(a! b! c! d!)

    • Thus the likelihood equation is


    Homozygosity mapping using lod score method


    Homozygosity mapping using lod score method

    • Hence Likelihood is: map units between A and B)

    • Likelihood needs to be calculated between each value of R i.e. 0.01 – 0.5.


    Step iii and iv
    STEP III and IV map units between A and B)

    • Data from several families are added and compared to get a good estimate of R

    • Standardization of L value which means calculation of Odds Ratio (OR)

    • Then Logarithm of OR is taken which is LOD score

    • LOD scores from various families are added (this is like AND rule for two events i.e. Family 1 AND family 2 ---- Both occurring)


    Homozygosity mapping using lod score method

    • A total LOD score for some R value of 3 is considered proof of linkage of two genes

    • In our example,

      • Odds Ratio = L0.20 / L0.50

        = 0.0301 / 0.00695

        = 4.331

      • LOD score = Log10 4.331 (Log10 OR)

        = 0.637

    • It is evident from this score that data from several families of this size is needed to reach a lod score of 3.0 as a proof of linkage.