DNA Markers

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DNA Markers

• October 12, 2007
• Ian Wilson

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Objectives

• Describe the techniques and theory behind
• RFLP analysis
• VNTR analysis
• SNP arrays
• Be able to solve problems using these techniques

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Avenues of attack

• Look at differences in where restriction enzymes
  cut
• Look at differences in lengths of VNTRs
• Direct sequence analysis to identify differences
  between individuals

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RESTRICTION FRAGMENT LENGTH POLYMORPHS
DIFFERENCES IN VNTR LENGTH
Southern blot
PCR
Restriction Digest/Southern blot
PCR
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RFLP

• Restriction-fragment length polymorphism
• What does this mean!!!
• Two main methods of analysis
• Radiolabeled oligonucleotide
• PCR based

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Restriction Enzymes

• Cut at 4-12 base pair sequences of dsDNA
• Can generate either sticky or blunt ends
• How often will an enzyme encounter a given
  sequence?
• What cuts more frequently, an 8-cutter or a
  4-cutter? How much more frequently?

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Restriction enzymes
Sticky
Blunt
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Back to RFLP

• For Southern blot analyses
• Genomic DNA is digested (cut up) with restriction
  enzyme
• Run on agarose gel for size separation smaller
  fragments run farther
• Transferred to a nitrocellulose or nylon membrane
  (named for Edwin Southern)

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RFLP ctd

• Use a single stranded oligonucleotide to probe
  membrane
• Single stranded oligonucleotides will find
  complementary sequences and bind to them
• Make radiograph (use X-ray film), and bands will
  show up where the probe has bound
• Alternatively, chromogenic dyes may also be used.

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Cut and uncut DNA
Lane 1 - MW marker Lane 2 - Uncut gDNA Lane 3 -
Cut gDNA Lane 4 - Uncut gDNA Lane 5 - Cut gDNA
Longer
Shorter
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PCR-RFLP

• RFLP analysis can be performed on PCR products
• PCR is performed and the PCR products are
  digested using the restriction enzymes
• Digested products are run on an agarose gel for
  size separation
• DNA in agarose gels can be visualized with EtBR
• What is PCR?

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PCR products

• Products of PCR reactions are usually very
  specific
• This results in many copies of the same sequence,
  and shows up on a gel as a concentration of DNA
  at a certain size

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What does this look like?
1 - pcr region of interest 2 - digest with
enzyme 3 - run on gel
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So how do we work this RFLP stuff?

• Design oligonucleotides and PCR primers carefully
• When interpreting gels, dont forget each person
  should have 2 alleles (one maternal, one paternal)

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VNTR fingerprinting

• Exploits inter-individual diversity in VNTR
  elements
• Remember what VNTR stands for?
• These are typically inherited in a mendelian
  fashion.

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How can we exploit this?

• Ideas?

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Two ways to analyze VNTRs

• Using radiolabeled oligos and restriction enzymes
  that dont cut the VNTR (if you know its
  sequence)
• Using primers that amplify around the VNTR we can
  check how many tandem repeats there are.

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Using Southern blots
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Again
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Using PCR
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What weve covered

• RFLP
• Southern blot
• PCR
• VNTR
• Southern blot
• PCR

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SNPs

• Single nucleotide alterations spread throughout
  the genome
• Over 4 million mapped
• Must occur in more than 1 of pop

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How can we assess these?

• Hint! Weve already talked about it

RFLP analysis would work!
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Moving to the future

• Microarrays allow us to look at many SNPs at once
  (500,000)

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