13' Positional Cloning and Gene Mapping

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13. Positional Cloning and Gene Mapping
a). General mapping strategies i). Genetic
mapping ii). Physical mapping b). RFLP linkage
analysis i). RFLPs linked to disease
genes ii). Predictive value of linked
RFLPs c). Gene isolation with linked RFLPs i).
Chromosome walking ii). Criteria for
identifying a gene d). Physical mapping of a
gene i). Cytogenetic analysis ii).
Fluorescence in situ hybridization (FISH)
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• General mapping strategies
• genetic mapping
• definition the ordering of genes on chromosomes
• according to recombination frequency
• genetic mapping may sometimes be required
• first, before probes can be found to use in
• physical mapping
• physical mapping
• definition the determination of physical
  distances
• between genes (in base pairs of DNA) using
• cytogenetic and molecular techniques
• physical mapping is used to ultimately pinpoint
• the gene of interest

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• RFLP linkage analysis
• RFLPs provide useful markers for all of the
  human chromosomes
• disease genes can be mapped by searching for
  linkage between an
• RFLP and the disease phenotype

RFLP probe
disease gene
A
normal gene
a

• the A/a polymorphism is linked to the
  disease-causing gene

AA Aa aa
AA homozygous for A Aa heterozygous aa
homozygous for a

• In a dominant disease, AA and Aa would
• be affected
• In a recessive disease, AA would be
• affected and Aa would be a carrier

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• If it is known on what chromosome (and where on
  that
• chromosome) the RFLP is located, the location of
  the
• disease gene can be determined - this maps the
  gene

• In addition, establishing linkage between an
  RFLP and
• a disease phenotype will allow
• prediction of those at risk for a disease
• isolation of genes by positional cloning

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• linkage of the PKD1 (polycystic kidney disease)
  gene
• to locus 2 but not locus 1
• (locus 1 is too far away on the same chromosome
  to show linkage)

a
A
Locus 1
B
PKD1
b (wild type)
c
C
Locus 2
A
A
a
A
a
B
b
b
b
B
C
c
c
c
C
(only the paternal chromosomes are shown)
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Linkage allows predictions to be made
RFLP probe
normal gene
A
disease gene
a

• the A/a polymorphism is linked to a gene that
  causes a genetic disease
• if the disease gene has not yet been identified
  or isolated, the established
• linkage can nevertheless have predictive value
• first, determine which allele cosegregates with
  the disease phenotype
• within the family being examined -- because of
  the potential for
• recombination to occur it is possible that
  either A or a is linked
• to the disease gene
• second, test the subject (e.g., prenatal or
  presymptomatic) to determine
• its genotype

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RFLP probe
normal gene
A
X
recombination?
disease gene
a

• the test subject is determined to have the
• same genotype as its sibling and therefore
• can be predicted to get the disease
• the prediction must be qualified,
• however, because of the
• possibility of recombination
• between the polymorphic
• marker and the disease gene
• the frequency of recombination
• and therefore the reliability
• of the marker is dependent
• on the distance between the
• two sites

affected child
test subject
mother
father
Aa Aa aa aa
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Linkage analysis for PKD
PKD Upper band in Southern blot is linked to
PKD
Southern blot

A
a
Upper band is diagnostic
Lower band is diagnostic

recombinant
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• How does one isolate a gene for an inherited
  disorder?
• There are three options
• Start with a candidate protein
• DNA protein
• Start with a candidate mRNA
• DNA mRNA
• Direct positional cloning
• DNA

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• Gene isolation with linked RFLPs (positional
  cloning)
• isolation of a genomic clone using the probe for
  the RFLP should allow
• the isolation of the downstream gene by
  chromosome walking

RFLP probe
normal gene
A
disease gene
a
isolate genomic clone by screening a genomic
library with the probe
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• partial restriction enzyme digestion allowed
  cloning of
• overlapping fragments starting with an RFLP probe

can be a large distance (several cM)
starting point (RFLP)
gene
new probe
gene being sought
etc.

• chromosome walking involves
• isolation of a genomic clone
• isolation of the downstream end of the clone
• use of this end piece to screen the genomic
  library
• for clones that are further downstream
• repeat the process and walk down the
  chromosome
• chromosome walking is accelerated by use of
  genomic libraries
• cloned in cosmid or YAC vectors that handle much
  larger pieces
• of DNA as compared to bacteriophage

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• Criteria for positive identification of a disease
  gene
• Presence of a mutation
• comparison of normal and affected individuals is
  necessary
• multiple normal and affected family members are
  examined
• candidate mutation must be found only in
  affected individuals
• Mutation can disrupt gene function
• mutation must be one that would disrupt normal
  information flow
• must distinguish between benign polymorphisms
  and mutations
• can include large deletions, frameshift
  mutations, splicing
• mutations, nonsense (stop codon) mutations
• Abnormal gene function explains pathogenesis
• gene must normally be expressed in the affected
  tissues
• expression is analyzed by
• Northern blotting - for mRNA expression
• Western blotting - for protein expression
• function of the gene is consistent with the
  physiological

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Southern blotting procedure
human genomic DNA (isolated from
many cells)
-

• gel electrophoresis
• of the DNA fragments
• gel will separate DNAs
• according to size

• restriction enzyme
• digestion

millions of DNA fragments

• hybridize membrane with
• a 32P-labeled DNA probe
• probe will base pair with the
• complementary DNA strands

• denature
• DNA into
• single-
• strands
• transfer
• DNA fragments to
• nitrocellulose membrane

• expose membrane to X-ray film
• develop film to visualize DNA band

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Northern blotting procedure

• human cells (eg. liver, brain, etc.)

-

• gel electrophoresis
• of the mRNAs
• gel will separate mRNAs
• according to size

• isolate total
• cellular mRNA

thousands of intact mRNA molecules

• hybridize membrane with
• a 32P-labeled DNA probe
• probe will base pair with the
• complementary mRNA strands

• transfer
• mRNAs to
• nitrocellulose membrane

• expose membrane to X-ray film
• develop film to visualize band

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Western blotting procedure

• human cells (eg. liver, brain, etc.)

-

• gel electrophoresis
• of the proteins
• gel will separate proteins
• according to size

• isolate total
• cellular protein

thousands of intact protein molecules

• react membrane with
• an antibody probe
• probe will bind to the
• protein

• transfer
• proteins to
• a membrane

develop blot to visualize band
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Criteria for positive identification (continued)

• Genotype-phenotype correlation
• different mutations give rise to different
  disease severities
• comparisons between families is made
• Correction of the defect cures the disease
• needs a gene therapy approach
• this is the ultimate test that the disease gene
• has been identified

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• What further can be done with a gene isolated by
  positional cloning?
• Map the gene to a chromosomal region to
  determine if it
• correlates with a site containing a known
• cytogenetic abnormality
• Cytogenetic analysis
• Fluorescence in situ hybridization (FISH)
• Look for abnormalities in the DNA sequence to
  determine
• if the gene is mutated, to identify the specific
  mutation,
• and to screen for mutations in other individuals
  
• Southern blotting
• DNA sequencing
• Allele-specific oligonucleotide (ASO) analysis
• PCR analysis

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• Cytogenetic analysis
• deletion mapping

1
2

• deletions 1-6 that all give rise to a
• particular disease phenotype map the
• locus to a narrow region between the
• right end of del4 and the left end of del5

3
4
5
6
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• Fluorescence in situ hybridization (FISH)
• DNA probe labeled with a fluorescent tag is
  hybridized to denatured
• metaphase chromosomes (resolution is 1-2 million
  bases)
• used to determine
• whether there is missing chromosomal material

normal chromosome
two different chromosomes with deletions

• the probe hybridizes to the normal chromosome,
  but not
• to either deletion chromosome, indicating that
  the gene is
• localized to the small deleted area common to 4
  5
• between the dotted lines

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• Cytogenetic analysis
• translocation breakpoint analysis

normal chromosomes
breakpoint chromosomes

• breakpoints that give rise to a particular
• disease can pin-point the disease gene if they
• lie within (and therefore disrupt) the gene

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• Fluorescence in situ hybridization (FISH)
• DNA probe labeled with a fluorescent tag is
  hybridized to denatured
• metaphase chromosomes (resolution is 1-2 million
  bases)
• used to determine
• localization of the gene being mapped

• the probe hybridizes to both breakpoint
  chromosomes
• indicating that the breakpoint is within the gene

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http//www.ncbi.nlm.nih.gov/disease/
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Tumor Suppressor Gene (MLL) on Chromosome 11 and
on the Nucleus, 1997 By Gary Schneider
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• Learning objectives
• understand the difference between genetic
  mapping and
• physical mapping
• understand the relationship between genetic
  linkage analysis
• and RFLP linkage analysis
• know the methods for RFLP linkage analysis
• understand what genetic and medical knowledge
  can be derived
• from RFLP linkage analysis
• understand the limitations of RFLP linkage
  analysis
• understand the strategy and methodology for
  positional cloning
• know the criteria for positive identification of
  a disease gene
• understand how physical mapping interrelates
  with
• cytogenetic analysis