DNA Fragment Analysis

DNA Fragment Analysis What shows have you seen on TV that use or talk about DNA evidence?

How is DNA really analyzed? Samples collected DNA isolated and extracted DNA is cut or amplified and sorted -Most common method is gel electrophoresis Results are analyzed

Collecting samples Must be collected carefully to prevent contamination Only a small amount needed

Isolating DNA You isolated DNA from various plant and animal tissues the other week Care must be taken to ensure DNA strand is not incomplete or damaged

Cutting or amplification of DNA If the sample is large you can use restriction enzymes to cut the DNA into small pieces

Restriction Enzymes Special enzymes that recognize specific sequences in DNA and cut it there Name comes from the bacteria isolated from Bacteria use them for defense

Restriction Enzymes Enzyme Organism from which derived Target sequence (cut at *)5' -->3' Ava I Anabaena variabilis C* C/T C G A/G G Bam HI Bacillus amyloliquefaciens G* G A T C C Bgl II Bacillus globigii A* G A T C T Eco RI Escherichia coli RY 13 G* A A T T C Hae III Haemophilus aegyptius G G * C C Hind III Haemophilus inflenzae Rd A* A G C T T Hpa I Haemophilus parainflenzae G T T * A A C Kpn I Klebsiella pneumoniae G G T A C * C Mbo I Moraxella bovis *G A T C Pst I Providencia stuartii C T G C A * G Sma I Serratia marcescens C C C * G G G SstI Streptomyces stanford G A G C T * C Sal I Streptomyces albus G G * T C G A C Taq I Thermophilus aquaticus T * C G A Xma I Xanthamonas malvacearum C * C C G G G

Restriction Enzymes ….TGG ….ACC CCAGATGGCG GGTCTACCGCTTAA AATTCGGCG… Eco RI Hae III ….TGG ….ACC CCAGATGGCG GGTCTACCGCTTAA AATTCGGCG… GCCGC…

Cutting or amplification of DNA If the sample is large you can use restriction enzymes to cut the DNA into small pieces If sample is small copies of portions can be made Polymerase Chain Reaction or PCR

https://fp.auburn.edu/asim/State BIOLOGY/Bio Labs/B8EleDye/Teacher/PCR_reaction.swf

Sorting fragments Process called gel electrophoresis Gel made from agarose which is semi-solid DNA fragments sorted by Charge (DNA is negatively charged) Size (base pairs) Shape

Sorting fragments DNA fragments sorted by Charge (DNA is negatively charged) Size (base pairs) Shape How do you think these factors affect movement?

Electrophoresis (-) (+) DNA fragments Negative electrode Positive electrode (+)

Sorting fragments Small fragments travel further than large fragments Bands are formed by large numbers of similar sized fragments Standard or ladder is added to compare fragment sizes

Analysis Similar bands indicate similar DNA Remember where your DNA comes from Mom and Dad Multiple bands are needed for positive identification

Review DNA fragments can be used to identify individuals DNA is isolated, cut or amplified, and finally sorted by electrophoresis DNA bands can then be analyzed