Kinship DNA Fingerprinting Simulation Grab the packet from the front table and begin reading.

Overview 3.4 billion base pairs in human genome. Only 5% code for proteins. Function of other 95% of genome (non- coding DNA) is unknown.

DNA Fingerprinting The non-coding DNA is key to determining identity via DNA Fingerprinting. Why? –It is less conserved than coding DNA, so it is more likely to vary from person to person.

Polymorphisms Differences in the non-coding DNA from person to person are known as polymorphisms. Polymorphisms are passed down from generation to generation in a Mendelian way. Focusing on polymorphisms in different regions allows scientists to compile DNA “fingerprints” of individuals.

Polymorphisms Polymorphisms occur in regions of non-coding DNA called minisatellites. The minisatellite regions are composed of identical DNA sequence blocks repeated in tandem. However, the number of repeats varies from person to person. This type of polymorphism is called a “variable number of tandem repeats” or VNTR.

VNTRs are often flanked by restriction enzyme sites that can be utilized to create different length fragments containing the VNTR regions. The DNA sequence variations in the fragments are termed “restriction fragment length polymorphisms” or RFLPs. (often pronounced “rif-lip”

How to create a DNA profile: 1.) DNA is extracted out of cells. 2.) The DNA is then cleaved using restriction enzymes. Restriction enzymes cut the DNA in specific, known spots. –Ex: the enzyme PvuII recognizes the sequence 5’-CAGCTG-3’. Each time the enzyme recognizes this sequence, it breaks the DNA between the G and the C on both strands. This is called a blunt cut. (See Board).

Another example: –The enzyme EcoRI recognizes the sequence 5’-GAATTC-3’ and cuts between the G and A on both strands. This is called a staggered cut. (See board). 3.) Once the DNA is digested, it is subjected to gel electrophoresis.

Gel electrophoresis: A process of separating DNA into bands based on molecular weight (length) using electricity. DNA fragments are placed into the gel (at the (-) terminal and the current is turned on. Because DNA is negative, it will migrate through the gel towards the (+) terminal. –The speed of the movement is directly related to the molecular weight (length) of the DNA fragment.

Gel electrophoresis: Once finished, the gel is stained so that the DNA can be seen. DNA markers are commonly used in gel electrophoresis. –The markers are fragments of DNA of known length that distribute through the gel in a predictable pattern. –These are used to determine the unknown length of the DNA fragments you are testing.

Southern Blotting Problem with gel electrophoresis is when finished, you still cannot discern the specific VNTR regions (b/c the bands overlap in the gel). A solution is Southern Blotting, where probes are used.

Southern Blotting Probes are short sequences of single stranded DNA that are complimentary to a sequence of interest. Probes are modified to contain radioactive ions or fluorescent molecules that can be used to visualize the DNA. In DNA fingerprinting, these modified probes are targeted to specific VNTR polymorphic loci by having a base pair sequence that is complementary to part of the VNTR region.

Southern Blotting Before probes can be added, the DNA must be removed from the gel and transferred onto a thin membrane of nylon or nitrocellulose.

Forensics Most forensic labs today, instead of analyzing the VNTR regions of minisatellite DNA, examine short tandem repeats (STRs) of noncoding microsatellite DNA. STRs are short repeated units only 2-5 nucleotides long. The concept of human identification is the same with STRs, the approach is just different. Instead of Southern Blotting and probing, the labs use PCR to amplify the multiple STR regions, followed by DNA sequencing with fluorescently labeled nucleotides to determine the number of repeats in each STR allele.

Today’s activity You will simulate DNA fingerprinting to determine whether a certain parent is the parent of a particular child. A case study is presented in your student packet.

What each group of 4 students needs: 74 white 4-way beads 6 red 2-way beads 68 purple 2-way beads 24 orange 2-way beads 24 yellow 2-way beads 14 blue 2-way beads 12 green 2-way beads 40 white connectors 1 plastic container cup 2 paper gels 2 sheets of tracing paper 2 acetate sheets 1 black dry-erase marker