7.1 Using gene sequencing

Question 1

What is a genome?

Answer 1

all of the genetic material in an organism

Question 2

What does PCR stand for and do?

Answer 2

polymerase chain reaction
it amplifies a DNA sample - making multiple copies of it

Question 3

What are the 3 steps of PCR?

Answer 3

Denaturation
Annealing
Extension

Question 4

How does PCR amplify DNA samples?

Answer 4

1. DNA strands heated to 96oC to separate the strands
2. primers added to 2 separate strands
3. temp cooled to 50-65oC to help primers bind to DNA
4. primer attachment signals to a polymerase where to start DNA synthesis
5. 2 polymerase molecules attach to 2 primers on the 2 DNA strands. As they move along, temp goes up to 72, they create new complementary DNA

Question 5

What are the uses of PCR?

Answer 5

• to predict the amino acid sequence of proteins and possible links to
  genetically determined conditions, using gene sequencing.
• in forensic science, to identify criminals and to test paternity, using DNA profiling.

Question 6

What is a terminator base

Answer 6

modified versions of the 4 nucleotide bases that stop DNA synthesis
once the terminator base has incorporated no more bases can be added

Question 7

Describe the process of DNA sequencing?

Answer 7

1. DNA strands are chopped into smaller pieces
2. Double strands are separated to give single strands
3. PCR amplifies DNA sample for analysis
4. Labeled terminator bases added to
   single stands of DNA. Each terminator base has a fluorescent tag in a specific colour
5. Coloured tags enable the sequence of bases to be read rapidly by an automated system

Question 8

How can DNA sequencing be used to predict the amino acid sequence of proteins?

Answer 8

• Because genetic code is universal - you can recognise start/stop codons
• Base pairs can be analysed to work out which amino acids will be joined together to form a protein

Question 9

How can DNA sequencing be used to identify possible links to genetically-determined conditions?

Answer 9

• Many diseases are result of inherited gene variants
• By sequencing genes you can find out mutations associated with different genes and develop new targeted therapies

Question 10

What is the process of gel electrophoresis?

Answer 10

Fragments are placed in wells in agarose gels and dyed with ethidium bromide so they fluoresce under UV light.

A current is then applied to the gel. DNA is negative and fragments of different
sizes move at different speeds according to mass so ‘bands’ appear.

Question 11

What is Southern Blotting?

Answer 11

1. Gel placed in alkaline solution to denature DNA
2. Blot is created when nylon filter picks up DNA from the gel
3. Single stranded DNA probe with radioactive label is added to the filter
4. The probe hybridizes with its unique target sequence on the denatured DNA - which can then be identified.

Question 12

Describe the process of DNA profiling?

Answer 12

1. DNA strands are cut into fragments using restriction endonucleases
2. These fragments are separated and visualised using gel electrophoresis
3. Southern blotting occurs - alkaline buffer solution added to the gel, nylon filter placed over it - to draw the solution containing DNA fragments from gel to filter
   and the solution is denatured. Fragments visible as ‘blots’.
4. Gene probes (labelled complementary sequences that fluoresce or are radioactive) are added and bind with DNA (hybridization)

Question 13

How is DNA profiling used to identify criminals and paternity?

Answer 13

(talk about the bars and how they match up - use exam question answer)

Question 14

What is an exon?

Answer 14

coding regions of DNA - the genes

Question 15

What is an intron?

Answer 15

large, non-coding regions of DNA that are removed before mRNA is translated into proteins