7.1 Using gene sequencing

Question 1

what is the genome

Answer 1

the total of all the genetic material in an organism

Question 2

where is the dna in the following
- prokaryotes
- eukaryotes
- green plants

Answer 2

• prokaryotes: cytoplasm, in the main chromosomes and the plasmids
• eukaryotes: nucleus, mitochondria
• nucleus, chloroplasts

Question 3

how much of our DNA is coding DNA

Answer 3

less than 2%

Question 4

what is the name of:
- coding DNA
- non coding DNA

Answer 4

• coding: exons
• non coding: introns

Question 5

where is DNA profiling used:

Answer 5

• court of law
• paternity cases

Question 6

what does PCR stand for

Answer 6

• polymerase chain reaction

Question 7

what is DNA amplification

Answer 7

• when a tiny sample of DNA is increased using PCR to produce a large enough sample to analyse

Question 8

why is thermus aquaticus (Taq) used in PCR

Answer 8

• DNA samples need to be heated to 90-95 degrees to separate the 2 strands
• this destroys DNA polymerase
• enzymes in bacterium evolved to live in extreme conditions (hot springs)

Question 9

Describe the process of PCR

Answer 9

1. DNA sample is mixed with Taq, DNA polymerase, primers and a good supply of nucleotide bases and buffer
2. solution is placed in PCR machine
3. mixture heated to 90-95 degrees, this causes the strands to separate, hydrogen bonds break
4. mixture is cooled to 50-55 degrees, primers bind to DNA strands
5. mixture heated to 72 degrees, optimum temp for Taq to build complimentary DNA strands
6. steps are repeated 30 times

Question 10

describe the process of DNA sequencing

Answer 10

1. DNA strands are chopped into smaller pieces by endonucleases
2. double strands are separated to give single strands
3. PCR is used to amplify DNA
4. terminator bases are added, each ahs a different fluorescent colour
5. coloured tags enable sequence of bases to be read rapidly in an automated machine

Question 11

How can DNA sequencing be used

Answer 11

• can be used to determine the protein produced from any particular gene
• gives scientists a better understanding of human disease

Question 12

how does DNA sequencing help scientists gain a better understanding of disease

Answer 12

• helps identify faulty genes, and see which bases have changed and understand how changes in bases affect the protein produced, and how this results in the symptoms of the condition

Question 13

what DNA is used in DNA profiling

Answer 13

• introns are the region of chromosomes used in DNA profiling

Question 14

what is the exception of DNA profiling

Answer 14

• identical twins

Question 14

what are the steps of DNA profiling

Answer 14

1. extracting, purifying them amplifying DNA using PCR
2. Cutting DNA into smaller fragments (DNA restriction)
3. Separating DNA fragments by size (DNA gel electrophoresis)
4. separated DNA fragments are transferred to membrane (southern blotting)
5. Visualising the presence of s specific sequence (DNA hybridisation)

Question 14

describe the type of sequences within introns

Answer 14

• short sequences that are repeated many times to form micro satellites and mini satellites
  mini - 10-100 base sequence
  micro 2-6 base sequence
• they appear in the SAME SECTIONS of each pair of homologous chromosomes
• the number of repeats on each satellite vary from person to person

Question 15

describe the process of DNA extraction

Answer 15

• cells are broken up using pestle and mortar
• cells are lysed using a detergent that disrupts the plasma membrane
• cell contents is treated with protease and RNAase, to destroy proteins and RNA
• cell debris is pelleted in a centrifuge, liquid containing DNA is transferred into a new tube
• DNA is precipitated with ethanol, and forms viscous strands that can be spooled on a glass rod

Question 16

what is an endonuclease and what does it do, and what process is it used in

Answer 16

• they are restriction enzymes
• cut DNA ast restriction sites, and each is specific to each site
• protect us from viruses by cutting the DNA of viruses

Question 17

describe the process of DNA gel electrophoresis

Answer 17

• DNA fragments are placed in wells in agarose gel medium in a buffering solution, with known DNA fragments
• The gel contains a dye that binds to DNA fragments
• electric current is passed through the apparatus
• DNA fragments move towards the anode, move at different rates according to their size
• plate is placed under short UV light

Question 17

describe the process of southern blotting

Answer 17

• alkaline buffer solution is added to the gel
• nylon filter paper is placed over it
• alkaline solution denatures the DNA fragments so the strands separate and the bases are exposed
• DNA is covalently bound by UV cross linking

Question 17

what occurs during gene hybridisation

Answer 17

• Large quantities of gene probes are added to the filter and bind with complementary DNA strands
• Each probe is labelled with a fluorescent molecule or a radioactive isotope

Question 18

what is a gene probe

Answer 18

• a short DNA sequence that is complementary to the sequences that are being sought

Question 19

how do scientists identify individuals in forensic science

Answer 19

• gene probes are used to pick out short tandem repeats (microsatellite regions)
• the more microsatellites used, the more accurate it will be
• the more sites that are examined, the more likely it is that a different combination would have been inherited from 2 different parents
• a match on 11 or more sites is counted as reliable in court

Question 20

how are individuals identified in paternity testing

Answer 20

• specific microsatellite markers are used to make the matches
• the child would have some microsatellites from the mother and some from the father and some don’t come from either the mother or the father