University:
California State University, Northridge
Course:
BIOL 360 | Genetics
Academic year:

2007
Views:

99

Pages:

16
Author:

Maribel Carr

DNA Fingerprinting • Unless they are identical twins, individuals have unique DNA • DNA fingerprinting – The name used for the unambiguous identifying technique that takes advantage of differences in DNA sequence • The process of DNA fingerprinting begins by isolating DNA from – blood, semen, vaginal fluids, hair roots, skin, skeletal remains, or elsewhere 1 Polymerase Chain Reaction (PCR) • If there is only a small amount of DNA available for DNA Fingerprinting – augment the amount of DNA using a technique called PCR – PCR is doing DNA replication in a test tube 2 Polymerase Chain Reaction (PCR) • Like ALL DNA polymerases • Taq polymerase can only add to the 3’ end of an existing nucleotide 5’ 3’ 3’ 5’ and cool to anneal Template Primer Primer Template •A DNA primer that is complementary to the template is used to supply that 3’ end 3 DNA Fingerprinting • After we isolate the DNA and amplify it with PCR • Treat the DNA with restriction enzymes – cut DNA at specific sequences – Everyone’s DNA is different, so everyone’s DNA will cut at different sites • This results in different sized fragments • The different sized fragments are called restriction fragment length polymorphisms, or RFLPs • We can observe the different sized fragments in an experiment that separates DNA based on fragment size called Gel Electrophoresis 4 RFLP Analysis • Everyone has genetic sequences called variable number tandem repeats, or VNTRs – Everyone has different amounts of VNTRs – The VNTRs make the different sized RFLPs 5 Gel Electrophoresis • Fragments of DNA from restriction enzyme cleavage are separated from each other when they migrate through a support called an agarose gel – It is similar to the yummy food Jell-O gelatin – It is actually made out of some of the same ingredients • The size-based separation of Molecules of DNA separate based on size when an electric current is applied to an agarose gel – This is gel electrophoresis 6 Gel Electrophoresis 7 Gel Electrophoresis • The separated DNA fragments are then drawn out of the gel using a nylon membrane • The nylon membrane is treated with chemicals that break the hydrogen bonds in DNA and separate the strands • The single stranded DNA is cross linked to the nylon membrane – By heat or UV light • Incubate the nylon membrane with a radioactive probe of single stranded DNA complementary to the VNTRs 8 Gel Electrophoresis 9 Gel Electrophoresis • The radioactive probe shows up on photographic film – Because as it decays it gives off light – The light leaves a dark spot on the film • Different individuals have different patterns of bands – these make up the fingerprint 10 Gel Electrophoresis This Protocol is known as Southern Blotting 11 Southern Blotting DNA Fingerprinting • DNA fingerprints can be used to determine which bone fragments belong to which individual 13 DNA Fingerprinting • DNA fingerprints of children should be similar to the those of parents • DNA fingerprinting can show which individuals are the parents of specific children 14 Northern Blot Analysis • Northern blotting analyzes RNA much the same way that Southern blotting does DNA: – RNA is extracted from the cell, undergoes gel electrophoresis, and is bound to a filter. – Hybridization between bound cellular RNA and a labeled probe occurs. The sizes of the RNA fragments detected by the probe can be determined 15 Northern Blot Analysis • Northern blot analysis is used for determining: – The size(s) of mRNA encoded by a gene. Northern blots have shown that different mRNA species arise from the same region of DNA, suggesting differential use of promoters and terminators, and/or alternative mRNA processing. – Whether a specific mRNA is present in a cell type, and if so, at what levels. Gene activity is measured in this way, and RNA sampling is widely used to study development, tissue specialization, or the response of cells to various physiological stimuli. 16

DNA Fingerprinting