DNA profiling

Question 1

DNA extraction

Answer 1

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

Question 2

PCR (definition)

Answer 2

used to create multiple copies (amplify) a chosen segment of DNA

Question 3

uses of PCR

Answer 3

forensics, gene therapy, evolution

Question 4

PCR ingredients

Answer 4

target DNA, primers, DNA nucelotides, DNA polymerase

Question 5

PCR steps 1

Answer 5

the temperature is increased to around 95 degrees which causes breaking of the hydrogen bonds between the polynucelotide strands of the target DNA

Question 6

PCR steps 2

Answer 6

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

Question 7

PCR step 3

Answer 7

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

Question 8

Gel electrophoresis how it works

Answer 8

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

Question 9

distance travelled electrophoresis

Answer 9

smaller molecules travel faster and move greater distances, larger molecules travel at a slower rate and move a shorter distance

Question 10

southern blotting

Answer 10

a sheet of nylon is placed over the gel, DNA fragments stick to the sheet forming a profile

Question 11

DNA sequencing

Answer 11

the process of determining the nucelotide sequence of target DNA

Question 12

chain termination method

Answer 12

when PCR is carried out using dideoxynucelotides instead of deoxynucleotides

Question 13

ddNTPs

Answer 13

ddNTPs cannot form sugar phosphate bonds with other ddNTPs or dNTPs and this causes a termination of the growing polynucleotide in DNA replication

Question 14

DNA sequencing procedure

Answer 14

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

Question 15

electropherogram

Answer 15

A fluorescent compound is attached to the ddNTPs used in DNA sequencing.

Question 15

DNA profile

Answer 15

A DNA-based pattern composed of a series of bands corresponding to DNA fragments of different sizes.

Question 16

process of electropherogram

Answer 16

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.

Question 17

Tandem repeats

Answer 17

the repetitive sequences of bases in introns

Question 18

VNTRs

Answer 18

Variable Number Tandem repeats vary in length and chromosome location

Question 19

minisatellites

Answer 19

tandem repeat sequences between 10-70 bases in length

Question 20

microsatellites

Answer 20

tandem DNA sequences that are less than 10 base pairs in length

Question 21

VNTRs and gel

Answer 21

Larger VNTRs move slower, smaller VNTRs move faster

Question 22

DNA profiling procedure

Answer 22

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

Question 23

restriction enzymes

Answer 23

cut the sugar phosphate backbone at specific locations, restriction enzymes cut DNA at locations adjacent to a minisatellite or microsatellite

Question 24

restriction fragments

Answer 24

products of the restriction enzyme cutting the DNA

Question 25

probes

Answer 25

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.