Molecular Markers

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Molecular Genetics

COLLEGE

Last updated 3:49 PM on 10/4/26
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27 Terms

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<p>What is “Genetic Diversity?” </p>

What is “Genetic Diversity?”

The total number of genetic differences in a population


2
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What are some of the disadvantages of using morphological traits as a method of measuring DNA?

Dominance can mask diversity

environmental influences

<p>Dominance can mask diversity </p><p>environmental influences</p>
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What are the disadvantages of using whole genome sequencing to measure DNA?

More expensive

Requires complex data analysis

Need high quality DNA

<p>More expensive</p><p>Requires complex data analysis</p><p>Need high quality DNA</p>
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Why is using molecular markers a happy medium?

Relatively low cost

provide information on the loci not the whole genome

only need a small amount of DNA

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What are Molecular Markers?

A fragment of DNA that is associated with a specific location within the genome.


Can be used to identify a particular gene sequence

<p>A fragment of DNA that is associated with a specific location within the genome.</p><p></p><p>Can be used to identify a particular gene sequence</p>
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Molecular markers act as signposts along a gene, what does this help with?

  • Identifying genes for breeding/diagnostics

  • Discovering the location of unknown genes (by tracking the marker through generations)

  • Creating GENOME MAPS, these show the positions of features (genes + markers) along a chromosome


<ul><li><p>Identifying genes for breeding/diagnostics</p></li><li><p>Discovering the location of unknown genes (by tracking the marker through generations)</p></li><li><p>Creating GENOME MAPS, these show the positions of features (genes + markers) along a chromosome</p></li></ul><p></p>
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What are microsatellites/SSRs?

short repeats of DNA


1-6 bases repeated 5-20 times

<p>short repeats of DNA</p><p></p><p>1-6 bases repeated 5-20 times</p>
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What % of the human genome is made up of SSRs?

3%

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How are SRRs generated?

DNA replication slippage - elongates or shortens strands

<p>DNA replication slippage - elongates or shortens strands</p>
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What effects do SSRs have?

They affect DNA conformation, packaging and transcription

When it goes wrong it can lead to disease (cancer + neurological)

<p>They affect DNA conformation, packaging and transcription</p><p>When it goes wrong it can lead to disease (cancer + neurological)</p>
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What does using PCR on SSRs do?

Reveals the size and number of repeats in someones genome


“This person has heavier DNA so must have more repeats“

<p>Reveals the size and number of repeats in someones genome</p><p></p><p>“This person has heavier DNA so must have more repeats“</p>
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Why does it not matter how long the repeat is?

The flanking primers will be the same

<p>The flanking primers will be the same</p>
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What are the advantages of using microsatellites?

Lots of different lengths

reproducible results which can be transferred between labs

Multiplexing (More than one microsatellite tagging)

<p>Lots of different lengths</p><p>reproducible results which can be transferred between labs</p><p>Multiplexing (More than one microsatellite tagging)</p>
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What is a disadvantage of microsatellites?

The development of new SSRs can be time consuming


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How often does a Single Nucleotide Polymorphism occur?

Every 1000 bp

<p>Every 1000 bp</p>
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What are the three ways we can detect SNPs?

  • DNA sequencing - sequence DNA and compare it

  • Enzymes - If the change happens in a restriction enzymes active site then it cant cut the DNA (1 or two bands formed)

  • DNA hybridization - adding and detection of probes


<ul><li><p>DNA sequencing - sequence DNA and compare it</p></li><li><p>Enzymes - If the change happens in a restriction enzymes active site then it cant cut the DNA (1 or two bands formed)</p></li><li><p>DNA hybridization - adding and detection of probes</p></li></ul><p></p>
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What does DNA hybridization rely on?

Double stranded DNA can be heated to separate and a complimentary probe will bind to a target sequence

<p>Double stranded DNA can be heated to separate and a complimentary probe will bind to a target sequence</p>
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What are the 4 steps that all DNA hybridization requires?

  1. Isolate and prepare DNA - PCR

  2. Immobilisation of DNA on SOLID matrix - Southern blot/high density arrays

  3. Add labelled probe via hybridisation

  4. Detection of probe


<ol><li><p>Isolate and prepare DNA - PCR</p></li><li><p>Immobilisation of DNA on SOLID matrix - Southern blot/high density arrays</p></li><li><p>Add labelled probe via hybridisation</p></li><li><p>Detection of probe</p></li></ol><p></p>
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Whats the differences between southern blotting and high density arrays when immobilizing DNA?

  • Southern blotting -

    • Nitrocellulose filter is the solid support

    • Blotting paper is used to suck up DNA

  • High density arrays -

    • Glass slide is the solid support

    • Allows for a lot denser concentration (ideal for high throughput)


<ul><li><p>Southern blotting -</p><ul><li><p> Nitrocellulose filter is the solid support</p></li><li><p>Blotting paper is used to suck up DNA</p></li></ul></li><li><p>High density arrays -</p><ul><li><p>Glass slide is the solid support</p></li><li><p>Allows for a lot denser concentration (ideal for high throughput)</p></li></ul></li></ul><p></p>
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How are probes made and whats the difference?

  • In vitro DNA replication (label = radioactive nucelotides)

  • Synthetic DNA (Label = fluorescence)


<ul><li><p>In vitro DNA replication (label = radioactive nucelotides)</p></li><li><p>Synthetic DNA (Label = fluorescence)</p></li></ul><p></p>
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What does temperature affect in hybridisation?

Specificity of binding, if higher will be more specific

<p>Specificity of binding, if higher will be more specific</p>
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In RFLP assay 1 what is the starting material?

Genomic DNA that has been digested by a restriction enzyme

<p>Genomic DNA that has been digested by a restriction enzyme</p>
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Why in RFLP assay 1 do we have to do a southern blot with probes and not just stop at the agarose gel?

There are so many different lengths of DNA it would be too difficult to identify the DNA affected.

<p>There are so many different lengths of DNA it would be too difficult to identify the DNA affected.</p>
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What are the two disadvantages from RFLP assay 1?

  • Time consuming and needs a lot of DNA

  • A lot of background noise needed to sort through via hybridisation


<ul><li><p>Time consuming and needs a lot of DNA</p></li><li><p>A lot of background noise needed to sort through via hybridisation</p></li></ul><p></p>
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What the advantages of the RFLP assay 2?

  • quicker than southern

  • Less DNA needed

  • Less background noise - no hybridisation needed


<ul><li><p>quicker than southern</p></li><li><p>Less DNA needed</p></li><li><p>Less background noise - no hybridisation needed</p></li></ul><p></p>
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In RFLP assay 2 what is the starting material?

The SNP after its gone through PCR cycles —→ gets digested by restriction enzymes, produces 1 or 2 bands depending on is SNP present

<p>The SNP after its gone through PCR cycles —→ gets digested by restriction enzymes, produces 1 or 2 bands depending on is SNP present</p>
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What are the drawbacks of using restriction fragment length polymorphism assays?

  • need to know the DNA sequence in order to do the PCR