DNA profiling Flashcards
DNA extraction
the isolation of DNA from the cells of an organism, detergent and salt are added to dissolve the cell membrane ethanol is then added to precipitate the DNA
PCR (definition)
used to create multiple copies (amplify) a chosen segment of DNA
uses of PCR
forensics, gene therapy, evolution
PCR ingredients
target DNA, primers, DNA nucelotides, DNA polymerase
PCR steps 1
the temperature is increased to around 95 degrees which causes breaking of the hydrogen bonds between the polynucelotide strands of the target DNA
PCR steps 2
the temperature is decreased to around 55 degrees which allows primers to anneal to the seperated strands of target DNA through the formation of hydrogen bonds between complementary bases
PCR step 3
the temperature is increased to 72 degrees which is the optimum for the DNA polymerase enzyme. DNA polymerase extends the primers and creates new polynucleotide strands using DNA nucleotides
Gel electrophoresis how it works
DNA samples are loaded into wells on a plate of agarose gel that is attached to two electrodes positive and negative, DNA molecules are negatively charged and are attracted towards the positive electrodes
distance travelled electrophoresis
smaller molecules travel faster and move greater distances, larger molecules travel at a slower rate and move a shorter distance
southern blotting
a sheet of nylon is placed over the gel, DNA fragments stick to the sheet forming a profile
DNA sequencing
the process of determining the nucelotide sequence of target DNA
chain termination method
when PCR is carried out using dideoxynucelotides instead of deoxynucleotides
ddNTPs
ddNTPs cannot form sugar phosphate bonds with other ddNTPs or dNTPs and this causes a termination of the growing polynucleotide in DNA replication
DNA sequencing procedure
four test tubes are filled with many copies of the target DNA, all four deoxynucelotides (dNTPs) are transferred to each test tube, a different type of dideoxynucleotides is transferred to each test tube, primers and DNA polymerase enzymes are added to each of the four test tubes, the four test tubes are transferred to a thermocycler where DNA replication occurs, DNA replication is stopped whenever a ddNTP is added instead of a dNTP. This creates many incomplete copies of the template DNA each ending in a ddNTP. The contents of each test tube is transferred to four separate wells in agarose gel. The different fragments are separated using gel electrophoresis. The positions of the DNA fragments allow for the sequencing of the DNA molecule
electropherogram
A fluorescent compound is attached to the ddNTPs used in DNA sequencing.
DNA profile
A DNA-based pattern composed of a series of bands corresponding to DNA fragments of different sizes.
process of electropherogram
DNA fragments travel through the capillary tubes at different rates with smaller fragments moving at a faster rate.The fragments move through a beam of laser light which is absorbed by the fluorescent molecule attached to the ddNTP.The fluorescent molecule emits a light which enters a detector.
Tandem repeats
the repetitive sequences of bases in introns
VNTRs
Variable Number Tandem repeats vary in length and chromosome location
minisatellites
tandem repeat sequences between 10-70 bases in length
microsatellites
tandem DNA sequences that are less than 10 base pairs in length
VNTRs and gel
Larger VNTRs move slower, smaller VNTRs move faster
DNA profiling procedure
The DNA is extracted from sample, the extracted DNA is cut by restriction enzymes, restriction fragments are then seperated using electrophoresis, DNA is transferred to a nylon membrane, the nylon sheet is immersed in a solution of labelled DNA molecules called probes. X-ray film
restriction enzymes
cut the sugar phosphate backbone at specific locations, restriction enzymes cut DNA at locations adjacent to a minisatellite or microsatellite
restriction fragments
products of the restriction enzyme cutting the DNA
probes
the DNA sequence of the probe is complementary to the minisatellite or microsatellite under analysis. Probes generally contain a radioactive element, excess probes are washed away.
