Linkage Mapping Via Crosses

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Introduction– Definitions

Mapping: the experimental process of determining the relative locations of genes or other segments of DNA on individual chromosomes.

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Linkage Mapping

• It relies on the frequency of recombinant offspring to determine the distance between sites located along the same chromosome
• In eukaryotes, linkage mapping involves crosses among organisms that are heterozygous for two or more genes

Linkage Mapping Methods (ch.6)

• Allelic differences between genes were used to map the relative locations of those genes along a chromosome by conducting test crosses

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Molecular Markers Provide Sites for Mapping Experiments

• An alternative to relying on allelic differences between genes

• Regions of DNA that do not encode genes can be used as markers along a chromosome

• Molecular marker: Segment of DNA that is found at a specific site along a chromosome and has properties that enable it to be uniquely recognized using molecular tools

• Molecular tool

•  polymerase chain reaction (PCR)

• Gel electrophoresis

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• Molecular markers are polymorphic; within a population, they may vary from individual to individual

• Distances between linked molecular markers determine the outcome of crosses

      Purpose of Molecular Markers

• Used to determine the approximate location of an unknown gene that causes human diseases

• Identification of a particular marker in those who have the disease can indicate that the marker is close to the disease-causing allele

• Help researchers identify the location of genes involved in quantitative traits, such as fruit yield and meat weight, that are valuable to agriculture

 Common Types of Molecular Markers

RFLP: is a type of polymorphism that results from variation in the DNA sequence recognized by restriction enzymes. Scientists use these bacterial enzymes to cut DNA molecules at known locations.

AFLP: Same as RFLP nut the DNA sequence being used is amplified via PCR instead of isolating the chromosomal DNA

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SNP: are a type of polymorphism involving variation of a single base pair.  

• Ex. If you are looking at different genomes across individuals there will be certain nucleotides that are different from others. If I have a C and you have a C then that is an SNP. Some SNP’s do not hold much significance, but others confer human diseases (heart disease or diabetes)

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STS: Stretch of DNA that a PCR can easily detect

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Figure 22.4

• RFLPs: Restriction fragment length polymorphism

• The map shows the location of RFLP markers

• Numbers on the left indicate the locations of markers

• The numbers on the right are the distances in map units (mu)

• Ex. the RFLPs at map positions 16.3 and 40.2 on chromosome 2 are designated 551 and 251, and are 23.9 mu apart (40.2-16.3=23.9)

• The genes shown in red are a few important genes that are more known

• Atc4: encodes actin

• er: encodes erecta

• GH1: encodes acetolactate synthase

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Linkage Mapping Uses Molecular markers Such as Microsatellite

Microsatellites: short repetitive sequences that are abundantly interspersed throughout a species’ genome and tend to vary in length among different individuals

• They usually contain di-, tri-, tetra-, or Penta nucleotide sequences that are repeated many times in a row.

• Ex. The most common microsatellite encountered in humans is the dinucleotide sequence where n ranges from 5 to more than 50.

How do researchers identify a specific microsatellite within a chromosome?

• Shows the starting material is a sample that contains all of the chromosomes.
• Using primers complementary to the unique DNA sequences that flank a specific microsatellite allows that microsatellite to be amplified by PCR.
• The DNA fragments found in the two bands were made via PC, using primers that flank a particular microsatellite on chromosome
• The DNA fragment in the higher band is more extended because it has more repeat sequences than does the lower band.

If a pair of PCR primers copies a single site within a set of chromosomes, the amplified region is called a Sequence Tagged Site.

• A sequence-tagged site is a short DNA sequence that has a single occurrence in the genome and whose location and base sequence are known

When the DNA is collected from a haploid cell, an STS produces only a single band.

In a diploid species, an individual has 2 copies of a given STS. When an STS contains a microsatellite, the PC products may be identical and result in a single band on a gel if the region is the same length in both copies.

Figure 22.6

When microsatellites have length polymorphisms, researchers can follow their transmission from parent to offspring. PCR amplification of particular microsatellites provides a strategy in the genetic analysis of human pedigrees.

• Those that are not linked will independently assort from generation to generation. Those that are linked tend to be transmitted together to the same offspring.

• In a large pedigree, it is possible to identify cases in which linked microsatellites have segregated due to crossing over. The frequency of crossing over provides a measure of the map distance, in this case, between the different microsatellites.

  

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