DNA profilling

Question 1

genome

Answer 1

all the genetic material an organism contains (for eukaryotes it includes the DNA in the nucleus and mitochondria combined)

Question 2

exons

Answer 2

• the 20-25000 regions of DNA that code for proteins
• only make up about 2% of total DNA

Question 3

Introns

Answer 3

• the large non-coding regions of DNA that are removed from mRNA before it is translated into a polypeptide

Question 4

satellite DNA

Answer 4

• within introns, telomeres and centromeres are short sequences of DNA that are repeated many times

Question 5

minisatellite region/ VNTRs

Answer 5

• a sequence of 20-50 base pairs will be repeated from 50 to several hundred times
• occur at more than 1000 locations in the human genome and also known as variable number tandem repeats (VNTRs)

Question 6

microsatellite/STRs

Answer 6

a smaller region of 2-4 bases repeated only 5-15 times (short tandem repeats)

Question 7

where do satellites appear

Answer 7

• always appear in the same positions on the chromosomes, but the number of repeats of each mini or microsatellite varies between individuals as different lengths of repeats are inherited from both parents
• so only identical twins will have an identical satellite pattern but the closer related to someone, the more likely the patterns are similar

Question 8

who discovered these patterns in non-coding DNA

Answer 8

Alec Jeffreys

Question 9

DNA profilling

Answer 9

• producing an image of the patterns in the DNA of an individual
• used by scientists to assist in the identification of individuals or familial relationships

Question 10

stages of producing a DNA profile

Answer 10

• extracting the DNA
• digesting the sample
• separating the DNA fragments
• hybridisation
• seeing the evidence

Question 11

extracting the DNA in profiling

Answer 11

• DNA must be extracted from a tissue sample
• PCR (polymerase chain reaction) is used where the tiniest fragment of tissue can be enough to develop a profile

Question 12

digesting the sample in DNA profiling

Answer 12

• strands of DNA are cut into small fragments using restriction endonucleases
• different restriction endonucleases cut DNA at a specific nucleotide sequence (restriction/recognition site)
• all restriction endonucleases make 2 cuts, once through each strand of the double helix
• give scientists ability to cut DNA strands at defined points in the introns using mixture of restriction enzymes that leave the repeating units/satellites intact so the fragments at the end of the process include a mixture of mini and microsatellite regions

Question 13

separating the DNA fragments in DNA profiling

Answer 13

• cut fragments of DNA need to be separated to form a clear and recognisable pattern
• using electrophoresis which utilises the way charged particles move through a gel medium under the influence of an electric current
• the gel is immersed in alkali in order to separate the DNA double strands into single strands
• the single-stranded DNA fragments are then transferred onto a membrane by Southern blotting

Question 14

hybridisation in DNA profiling

Answer 14

• radioactive/fluorescent DNA probes are added in excess to the DNA fragments on the membrane
  DNA probes are short DNA/RNA sequences complementary to a known DNA sequence
• they bind to the complementary strands of DNA under particular conditions of pH and temperature
• DNA probes identify the microsatellite regions that are more varied than the larger microsatellite regions
• excess probes are washed off

Question 15

seeing the evidence in DNA profilling

Answer 15

• if radioactive labels were added to the DNA probes, X-ray images are taken of the paper/membrane
• if fluorescent labels were added to DNA probes, the paper/membrane is placed under UV light so the fluorescent tags glow
• the fragments give a pattern of bars (the DNA profile)

Question 16

what are DNA fragments put in in gel electrophoresis

Answer 16

• DNA fragments put into wells in agarose gel strips which contain a buffering solution to maintain a constant pH
• in usually the first and last wells, DNA fragments of known length are used to provide reference for fragment sizing

Question 16

how does DNA move in gel electrophoresis

Answer 16

• electric current passes through electrophoresis plate
• DNA fragments in the wells at the cathode (-ve) end move through the gel towards the positive anode at the other end
• due to negatively charged phosphate groups in DNA fragments

Question 17

what does the rate of movement of DNA through the gel in gel electrophoresis depend on

Answer 17

• the mass or length of the DNA fragments
• the gel has a mesh-like structure that resists the movement of molecules
• smaller fragments move more easily through the mesh than larger fragments
• so the smaller fragments move further than the large
• when the faster smaller fragments reach the anode (+ve) end of the gel, the electric current is switched off

Question 18

what happens in gel electrophoresis after the DNA moved

Answer 18

• gel is placed in alkaline buffer solution to denature the DNA fragments
• the 2 DNA strands of each fragment separate, exposing the bases

Question 19

Southern blotting

Answer 19

• the single stranded DNA are transferred to a nitrocellulose paper or nylon membrane which is placed over the gel
• membrane is covered with several sheets of dry absorbent paper, drawing the alkaline solution containing the DNA through the membrane by capillary action
• the single-stranded DNA fragments are transferred to the membrane as they are unable to pass through it
• transferred precisely the same relative positions as they had on the gel
• then fixed in place using UV light or heated at 80 degrees C