Topic 7.1 Using Gene Sequencing

Question 1

What is a genome?

Answer 1

Total of all genetic material in an organism

Question 2

PCR- Polymerase Chain Reaction

Answer 2

-A ‘DNA photocopier’
-Amplification of DNA in vitro (outside body)
-Without PCR there would be insufficient DNA for Human Genome Project

Question 3

How does PCR work?

Answer 3

1) DNA polymerase (duplicates DNA)
2) Primers x2 (piece of stranded DNA which is complimentary tot he specific target sequence at the 3’ end of each DNA replicated strand)
3) Free nucleotides to make amplified DNA
4) Original DNA strand needed to be replicated

Question 4

Amount of DNA made by PCR

Answer 4

PCR doubles the amount of DNA
eg. first cycle 2x2=4 fragments
second cycle 4x2=8 fragments

Question 5

PCR process summary: Denaturation (1)

Answer 5

• DNA is heated at 95°C
  -Hydrogen bonds between chains break.
  -Separate into 2 strands.

Question 6

PCR process summary: Annealing (2)

Answer 6

-Mixture cooled to 50-60°C
(Allows primers to anneal/attach to each 3’ end of each strand)

Question 7

PCR process summary: Extension (3)

Answer 7

• Heated to 72°C for DNA polymerase to attach nucleotides.
  -Heat tolerant DNA polymerase then replicates the region of DNA.
  -Takes longer for polymerisation of nucleotides.
• Repeated cycles of heating and cooling amplify this region of DNA by thermal cycler (30 times).

Question 8

DNA sequences

Answer 8

1) The DNA is cut into smaller pieces
2) Double strands are separated into single strands
3) PCR is used to amplify (replicate) the DNA
4) Terminator bases are added to the strands
5) The coloured tags allow the bases to be read quickly

Question 9

What are terminator bases?

Answer 9

They are modified versions of bases, they have a fluorescent tag attached.
A terminator base stops anymore bases from being added.

Question 10

Uses of DNA sequencing: Predicting amino acid sequences

Answer 10

Analysing the base pairs enables us to work out which amino acids will be joined together to form a protein as a result of information contained in DNA.

Question 11

Uses of DNA sequencing: Disease management

Answer 11

The knowledge we gain about the human genome will give us a better understanding of human disease.
There are small number of diseases that directly result from a mutation in a single gene.
DNA sequencing makes it possible to identify a faulty gene, see which bases have changed and understand how the changes in the DNA affect the protein produced.

Question 12

DNA profiling

Answer 12

DNA profiling refers to a process of analysing DNA for the purpose of identification.

Question 13

Genetic markers

Answer 13

Regions of DNA which usually vary between individuals.

Question 14

Short Tandem Repeats (STR)

Answer 14

A string of repeating nucleotide units (lots of different STRs throughout the genome)

Question 15

Steps in DNA profiling

Answer 15

1) Collect a DNA sample
(Obtained from any material which contains cells)
2) Extract DNA from sample
(Chemicals are added which break open the cells)
(DNA is separated from the other cell components)
3) Amplify STR fragments

Question 16

Mini satellite

Answer 16

10-100 base sequence, repeated 50 to several hundred times.

Question 17

Micro satellite (STR)

Answer 17

2-6 bases, repeated 5 to 100 times.

Question 18

Where are satellites found

Answer 18

Satellites are founding the same positions on each pair of chromosomes.
However, the number of repeats between individuals changes, depending on what you inherit from your parents.
Due to introns and the variation in the number of repeats, the chance of two individuals having the same pattern of DNA is extremely remote.

Question 19

Gel electrophoresis

Answer 19

1) DNA is ‘cut’ into pieces using enzymes
2) Electrical current separates the DNA pieces into gel
3) Separated DNA pieces are transferred to a membrane
4) Chemicals make the bands visible on the membrane

Question 20

What is a DNA probe?

Answer 20

• A short single stranded DNA that has a label attached to it making it visible.
• The probe is complementary to the DNA of the target allele.

Question 21

What is DNA profiling used for?

Answer 21

-In forensic science: to develop DNA in criminal investigations
-In paternity testing: to prove or disprove family relationships

Question 22

Describe the reaction mixture in the first stage of PCR

Answer 22

Contains:
-The DNA fragment to be amplified
-Primers that are complementary to the start of the fragment
-Free nucleotides to match up to exposed bases
-DNA polymerase to create new DNA

Question 22

What is DNA sequences?

Answer 22

Identify the base sequence of a DNA fragment.