Final Molecular Genetics

Definition of mapping:

Physical mapping is an approach to identify the physical location of a coding DNA (gene) sequence on the chromosome

Low resolution physical mapping:

1-10 map as the map scale

High resolution physical mapping:

1 to 100 bp as the map scale

Somatic hybrid cell preparation:

• Type of low resolution physical mapping

• conventional human-rodent hybrid cells

  • Human cells are fused with rodent cells in culture (polyethylene glycol, PEG)

  • The heterokaryon cells divide and form mononuclear cells which tend to lose human chromosomes randomly

  • A set of stable hybrid cells lines is established and each contains different human chromosomes

Monochromosomal hybrid cells:

• Part of low resolution physical mapping

  • Human cells are treated with colcemid to form microcells

  • Human microcells are fused with rodent cells to establish cell lines, each of which contains one human chromosome

Radiation hybrid cells:

• Part of low resolution physical mapping

  • Human cells are irradiated to break their chromosomes into smaller pieces randomly

  • The chromosome fragments are introduced into rodent cells

  • The frequency of two gene markers appearing in the same hybrid cells line is determined by the physical distance between them on the chromosome

Physical mapping of a gene who’s transcript (mRNA) sequence is known:

• Part of low resolution physical mapping

• Fluorescence in situ hybridization (FISH) - a large probe (40kb) is needed

  • Using FISH to define a translocation breakpoint

• Flow cytometry

  • Chromosomes are stained with fluorescent dyes

  • Flow cytometer sorts the chromosomes according to their fluorescence intensity which is determined by the DNA content (chromosome size) and nucleotide compositions

  • Monochromosomal hybrids may be used for better separation

High resolution physical mapping:

• 1 to 100 bp as the map scale

1. Smaller fragments may be obtained by partial restriction cleavages

2. These fragments (contigs) are assembled according to their overlapped regions

• chromosome walking

  • A specific DNA pore is used to screen a genomic library

  • Multiple positive clones must share the overlapping sequence (the probe sequence)

  • The positive clones are sequenced

  • A new probe may be made according to a unique sequence in one of the positives

  • Screening the genomic library again

  • YAC (yeast artificial chromosome) containing human chromosomal fragments may be used instead of total genomic DNA

Gene cloning is a process of identifying the gene for a particular ___

Phenotype

Gene cloning is a process of identifying the gene for a particular phenotype: it is divided into ___ approaches: ___________

Two; non-positional cloning and positional cloning

Positional cloning used when no sequence information is available but the ____ of the candidate gene on chromosomes is revealed through the pedigree analysis:

Approximate positioning

Positional cloning:

1. How is the approximate chromosomal location known?

   1. The rare recombinant in a pedigree:

   2. Linkage disequilibrium: The first mutation of a certain gene in an individual causes a genetic disease. The individual is the founder from whom the defective gene is inherited in the offspring. The gene allele close to the defective gene in the founder will be more associated with the defective gene in the offspring than other alleles in the population. Example is cystic fibrosis

2. Chromosome walking (crawling)

Non- positional cloning is used when some sequence information is available but the _____ of the gene is unknown:

Location

Non-positional cloning:

1. Identification of a disease gene through its protein product (hemophilia A)

   1. The biochemical basis of an inherited disease must be known first (the bleeding is due to the defective clotting factor VIII)

   2. The defective protein must be purified and sequenced

   3. The cDNA sequence is then deduced according to the deduced sequences

   4. The probe may be synthesized according to the deduced sequence

   5. The cDNA or genomic DNA libraries may be screened for the entire gene sequence

   6. Longer probes may be made and labeled with fluorescence for mapping the gene on a chromosome

2. Identification of a disease gene through its mRNA (phenylketonuria)

   1. Phenylketonuria is due to the lack of phenylalanine hydroxylase (PAH)

   2. PAH is extracted from rat liver and purified

   3. PAH is injected to animals for raising antibodies

   4. These antibodies are used to precipitate polyribosomes containing PAH and its mRNA

   5. The mRNA molecules are purified and reverse transcribed into cDNA

Mapping and identifying genes conferring susceptibility to complex diseases: the challenges

1. Although the disease is Mendelian, accurate diagnosis is not available

2. No apparent inheritance patterns are observable