DNA Typing

C. Gordeyev(Adapted in part from Larry J. Scheffler, Lincoln High School, 2009)

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Takes advantage of Mini/Microsatellites in DNA

• DNA that codes for proteins and has important functions is very similar in allhumans (conserved)

• Non-coding DNA is much more variable• Microsatellites are regions of tandem repeats of a pattern of

1-6 base pairs (ie - CAGCAGCAG)• Minisatellites are repeating patterns of 10-60 bp• The number of times that these patterns repeat in a given

area of DNA is highly variable in humans

http://www.nature.com/nm/journal/v11/n10/fig_tab/nm1005-1035_F2.html

Takes advantage of Mini/Microsatellites in DNA

• An individual will inherit a random half of their mother’s mini/micro satellites and a random half of their father’s (depending on how their chromosomes combine)

• The chances of 2 individuals (who aren’t identical twins) having the exact same pattern on mini and microsatellites are miniscule– Estimated by some as 0.253636 (1 in 5000 billion

billion)

• Therefore each DNA type is unique: a DNA fingerprint

Using Restriction Enzymes to study DNA

• One DNA molecule is far too long to be easily examined

• We digest (cut up) DNA with restriction enzymes

• Restriction enzymes will only cleave DNA at a specific sequence

http://www-math.mit.edu/~lippert/18.417/lectures/02_PartialDigest/Pictures/100000000000052F000003E8CBEA8CE5.jpg

Using Restriction Enzymes to study DNA• Each DNA molecule will only be digested

at certain locations that have this sequence

• Two non-identical DNA molecules will have different patterns of where they are digested

• This produces DNA fragments of different length

Gels: Sorting DNA Fragments by Length

• We load the DNA fragments into wells in one end of a polyagarose gel

• We run a current through the gel so that the end the DNA is inserted into is negatively charged and the opposite end is positively charged

• DNA is negatively charged, and migrates through the gel towards the positive end

http://img.youtube.com/vi/IWZN_G_pC8U/0.jpg

Gels: Sorting DNA Fragments by Length

• The smaller a DNA fragment is, the easier it is for it to wind its way through the gel to the other end

• The DNA fragments form a column of DNA Bands: smallest fragments closest to the positively charged end

http://www.cbs.dtu.dk/staff/dave/roanoke/fig5_33.jpg

BUT

• If we leave it like this there are so many bands that your column is one big blur

• We need some way of making this readable and useful

• We also want to look only at the areas that contain the mini/microsatellites

http://journals.prous.com/journals/servlet/xmlxsl/pk_journals.xml_summary_pr?p_JournalId=6&p_RefId=696541&p_IsPs=N

SO

• We transfer the DNA onto a membrane (nylon) and denature it (by use of enzymes) so our DNA is now single-stranded

• Single-stranded DNA will bind its complement strand very readily

Bands light up fluorescently• Then we make a piece

of single-stranded DNA that’s complimentary to the mini/microsatellite sequence, and attach a fluorescent molecule to it

• Where this binds to the gel, we see fluorescence

• This is where the mini/microstellites are

http://www.phac-aspc.gc.ca/publicat/ccdr-rmtc/05vol31/images/cdr3108_fig3.gif

Forensic DNA Analysis

• When many fluorescent probes are used to create many bands, this is called DNA Fingerprinting

• When only the 4-5 most indicative probes are used, we call this DNA Typing

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DNA Fingerprinting Process

DNA fingerprinting is a multistep process.

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Forensic DNA Analysis• Used to identify people Used to identify people

in criminal cases.in criminal cases.• Used to establish Used to establish

identity, paternity and identity, paternity and ancestry.ancestry.

• Used to study Used to study evolutionary changes evolutionary changes in speciesin species

• Used to ascertain Used to ascertain genetic disease genetic disease inheritance inheritance (Huntington’s disease)(Huntington’s disease)

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Forensic DNA Analysis

• DNA evidence is only DNA evidence is only as good as the person as good as the person performing the tests. performing the tests. Care must be taken to Care must be taken to guard against guard against contamination for legal contamination for legal evidence to stand.evidence to stand.

• Chain of evidence Chain of evidence rulesrules

• The more individual The more individual bands match up, the bands match up, the more valid the resultsmore valid the results

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