Topic 7 - Modern Genetics

Question 1

What is the Genome?

Answer 1

The genome is the total of all the genetic material in an organism.

Question 2

What are Exons?

Answer 2

Exons are the segments of DNA which contain information for the synthesis of proteins/peptide chains.

Question 3

What are Introns?

Answer 3

Introns are the segments of DNA which do not code for proteins/peptide chains.

Question 4

What does PCR stand for?

Answer 4

Polymerase Chain Reaction.

Question 5

How is PCR used in DNA profiling?

Answer 5

• PCR can Amplify small traces of DNA to create a large enough sample.
• (DNA profiling requires at least 1 microgram of DNA)

Question 6

What was the initial problem for scientists when trying to develop a way to replicate (amplify) DNA?

Answer 6

• DNA samples need to be heated to 90-95 ℃ to separate the strands (to make them available for replication).
• This temperature would destroy DNA polymerase from most organisms

Question 7

What is needed for a PCR?

Answer 7

• DNA sample (to be amplified)
• Taq DNA Polymerase
• Primers
• Supply of the 4 nucleotide bases
• Buffer
• PCR machine

Question 8

What are the three steps in a Polymerase Chain Reaction (PCR) ?

Answer 8

Step 1 - Heat a mixture of the DNA sample, Taq polymerase, buffer, free DNA nucleotides and finally primers at 90-95 ℃ for 30 seconds.

Step 2 - Cool the mixture to 50-55 ℃ for 20 seconds to let the primers bind to the DNA strands.

Step 3 - Heat the mixture again to 72℃ for at least a minute to let the Taq DNA polymerase build up complementary strands of DNA.

Question 9

What are each of the three steps in PCR called?

Answer 9

Step 1 - Separating the DNA strands

Step 2 - Annealing

Step 3 - Elongation

Question 10

What is the role of Primers in PCR?

Answer 10

• A pair of Primers will attach to each strand of DNA to mark the start points for the Taq DNA polymerase.
• In other words, primers highlight what section of the DNA is to be amplified.

Question 11

What is the role of Taq DNA Polymerase in PCR?

Answer 11

• Like any other polymerase, Taq Polymerase is involved in attaching DNA Nucleotides together to synthesise a strand of DNA.

Question 12

What is DNA sequencing?

Answer 12

DNA sequencing is the process of working out the order of bases in a strand of DNA.

Question 13

What is a terminator base?

Answer 13

• (In DNA sequencing) A Terminator base is a modified version of one of the 4 nucleotide bases.
• Terminator bases act as the the stop point for any further synthesis of DNA.

Question 14

How do Terminator Bases stop the further synthesis of DNA?

Answer 14

• Terminator Bases are modified so that they lack the -OH group (which is usually on Carbon 3 of the deoxyribose sugar).
• This results in the Terminator base being unable to bind with the phosphate group of another nucleotide and forming a phosphodiester bond.
• Therefore the strand of DNA stops extending.

Question 15

What are satellites? (DNA Profiling)

Answer 15

Satellites are short sequences of DNA bases that are repeated many times throughout the genome.

Question 16

What is the difference between Mini-satellites & Micro-satellites?

Answer 16

Mini-satellites have a longer base sequence (10-100 base pairs in a sequence) and are repeated more times throughout the genome.

Micro-satellites have shorter base sequences (2-6 base pairs in a sequence) and are repeated much less throughout the genome.

Question 17

What are VNTRs in DNA?

Answer 17

• Variable Number Tandem Repeats (VNTRs)
• Refers to the number of repeating sequences (satellites) found in DNA which differ in each individual.
• Unless in the case of Identical Twins.

Question 18

What are Restriction Endonucleases?

Answer 18

Restriction Endonucleases are a type of enzyme that cut up DNA at a specific sequence of bases called ‘Recognition Sites’.

Question 19

What is a Recognition site?

Answer 19

• A recognition site is a specific sequence of nucleotides in DNA which a Restricition Endonuclease (enzyme) recognises & digests.

Question 20

What do Specific Restriction Endonucleases do in DNA profiling?

Answer 20

• Specific Restriction Endonucleases are used to cut DNA in to fragments, whilst leaving VNTRs intact.

Question 21

Since Restriction Endonucleases don’t digest VNTRs, how does this create a specific DNA profile for an individual?

Answer 21

• Since VNTRs differ in length in each individual & Restriction Endonucleases don’t digest them, each individual will have a DNA profile consisting of different sizes of DNA fragments.

Question 22

What process separates the DNA Fragments based on size to create the DNA profile?

Answer 22

Gel Electrophoresis.

Question 23

How does Gel Electrophoresis separate DNA strands in terms of size?

Answer 23

• DNA molecules contain negatively charged phosphate backbones.
• The Gel Electrophoresis apparatus consists of a positive electrode on one side of an Agarose gel medium (DNA is on the opposite side in wells)
• The DNA strands will be attracted to this positive charge and will move towards the positive electrode.
• Larger DNA strands move slower and shorter DNA strands move faster.
• Therefore at the end of a certain time period (usually 45 minutes) the strands will have a been separated based on size.

Question 24

In Gel Electrophoresis, how do we make sure that the DNA strands don’t run off the end of the apparatus?

Answer 24

• A dye is added with the strands of DNA.
• This dye does not bind with the DNA but instead moves slightly faster than the DNA.
• Therefore when the dye reaches the end, we can stop the electric current in the apparatus to stop DNA strands running off the end.

Question 25

What is added to the DNA strands before Gel electrophoresis so we can Identify where the DNA strands are
at the end of Gel electrophoresis?

Answer 25

• A dye is added which binds to the DNA.
• This dye is fluorescent so that the DNA’s position can be observed at the end of Gel Electrophoresis using UV light.

Question 26

What is the name of the small holes DNA fragments are placed in during Gel electrophoresis?

Answer 26

Wells.

Question 27

What is the name of the medium used in Gel Electrophoresis?

Answer 27

Agarose Gel medium.