DNA L3 & 4 DNA Profiling, Southern Blotting : DNA Fingerprinting PDF

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DNA Profiling, Southern Blotting : DNA Fingerprinting...

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Lecture 3 & 4 DNA Profiling, Southern Blotting / DNA Fingerprinting [old method] What you need to know - What a pair of homologous chromosomes is - The human nuclear genome comprises 23 pairs of chromosomes: - - 22 pairs of homologous chromosomes and one pair of sex chromosomes - - Females have an XX pair of sex chromosomes - - Males have an XY pair of sex chromosomes - A pair of chromosomes comprises one chromosome from the mother and one from the father - Definition of a hypervariable region - There are two types of hypervariable region repeat unit - What a DNA fingerprint is - The principle of DNA fingerprinting - How to interpret DNA fingerprints - Hypervariable regions can mutate between one generation and another (i.e. are unstable) Key points - The old method of DNA fingerprinting relied on minisatellites and used Southern blotting DNA fingerprint/profiling A Prelude; Rudimentary Genetics: This was invented by Alec Jeffreys in 1985 in Leicester. It has many applications including; immigration disputes, paternity issues, murder cases and identification.

Principle behind DNA Fingerprinting: To exemplify this, we use a DNA schematic to show which part is maternal and which is paternal. A Hypervariable region is the place in which a specific sequence is repeated in tandem (one after another).The first block is the original which is then repeated. There are types of repeat

sequences; 1) Microsatellite or Short Tandem Repeats (STR): the length of sequence repeat of base pairs is between 2 and 4. Its SHORT 2) Mini satellites or Variable Number of Tandem Repeats (VNTRs): there are several 10s of base pairs, e.g. 20, 15, 30.

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On the diagram, the M represents the maternal chromosomes and the D represents the paternal. These are double strands of DNA. Primers are added to the region just before the hypervariable region and these primers have fluorescent probe molecules attached to them. The same primer, either forward or backwards, binds to the ‘same’ region on the M and D as the base sequence is the same on the maternal and paternal chromosome. One pair of primers amplifies 2 segments of DNA; one hypervariable of mother (M) and one of father (D). The result of this is 2 different bands.

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A locus is a region on a chromosome All schematics are of double stranded DNA M: maternal and P: paternal Repeat units are mini satellites Loci A,C,D may be on the same chromosome or different, it doesn’t matter, just somewhere on the genome The arrows represent cut sites Recognised by restriction enzymes These make cuts either side of the hypervariable region Its double stranded DNA that is cut

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For individual A, you can see from the gel electrophoresis that there are different sized fragments which migrate at different speeds. Each human (minus twins/clones) have different DNA fingerprinting so individuals A,B,C have different patterns

Application of DNA fingerprinting (1): this example will be of a paternity dispute. To solve this you would: 1. Get DNA by swabbing saliva cells 2. Obtain DNA fingerprint 3. Go along child’s results and match with mother 4. Go along child’s results and macth with father Sometimes one won’t match due to a mutation which we will look at shortly

Application of DNA fingerprinting (2): Another example is a murder case. Conclusions: suspect wasn’t the attacker and the same person carried out both attacks.

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DNA Fingerprinting & Profiling: Possibility of mutations: sometimes there are bands that don’t match up to the mother or father when looking at a DNA profile. This is called a mutation. This is due to the hypervariable loci being unstable so when you carry out PCR you get a stutter that deletes or inserts a repeat unit. For example, when we see a repeat unit being lost on the maternal chromosome. This will create a shorter hypervariable region that will migrate faster on the southern blot.

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