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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.
slide28

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
slide32

Sex limited trait…evidence

  • Crossed:
    • White eyed males (original) X F1 daughters…
  • 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
slide36

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
slide42

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%
slide45

Determine the phenotype of each cell in Punnet square

  • Add up the frequencies to get the total frequency of each offspring phenotype
slide46
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.
slide47

First define the terms for the observed family

    • 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
  • Multinomial theorem describing actual family: paqbrcsdmultiplied by a coefficientn! /(a! b! c! d!)
  • Thus the likelihood equation is
slide49

We have calculated phenotypic proportions for R = 0.20 (20 map units between A and B)

  • A family of 5 children has
    • 2 children with A_B_ phenotype
    • 1 with aaB_
    • And 2 with aabb
slide50

Hence Likelihood is:

  • Likelihood needs to be calculated between each value of R i.e. 0.01 – 0.5.
step iii and iv
STEP III and IV
  • 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)
slide52

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.
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