Manipulating Genomes

Question 1

How much of DNA does exons make up

Answer 1

2%

Question 2

What are satellites

Answer 2

In introns there are shorter sequences of DNA that are repeated many times these are satellites

Question 3

What is a mini satellite

Answer 3

A sequence of 20-50 bae pairs repeated from 50 to several hundred times

Question 4

How many times can mini satellites be found in the genome and what are they also called

Answer 4

Over 1000 locations on the genome and they are also called variable tandem repeats.

Question 5

What is a micro satellite

Answer 5

A smaller region of 2-4 bases repeated only 5-15 times also known as short tandem repeats (STRs)

Question 6

What is a feature of microsatellites and satellites

Answer 6

They always appear on the same position on the chromosome but the number of repeats of each mini or micro satellite varies between individuals, as different lengths are inherited from each parent.

Question 7

Who has identical satellite patterns

Answer 7

Identical twins

Question 8

How do you produce an image of the patterns of DNA

Answer 8

In DNA profiling

Question 9

What is the steps in DNA profiling

Answer 9

1. Extracting the DNA
2. Digesting the sample
3. Separating the DNA fragments
4. Hybridisation
5. Visualisation

Question 10

What is the process of extracting the DNA for DNA profiling

Answer 10

Polymerase chain reactions are used so that the tiniest fragment of DNA can give scientists enough data to develop a profile.

Question 11

What is the process of digesting the sample for DNA profiling

Answer 11

Strands of DNA are cut into small fragments using special enzymes called restriction endonucleases.

These give scientists the ability to cut DNA strands at defined points in the introns. SO they use a mixture of restriction enzymes that leave the satellites intact, so the fragments at the end include micro and mini satellites.

Question 12

What do different restriction enzymes do

Answer 12

They cut the DNA at a specific nucleotide sequence known as a restriction site or recognition site.

Question 13

What do the restriction enzymes do

Answer 13

They all make two cuts one through each strand of DNA in the double helix

Question 14

Why do the DNA fragments need to be separated

Answer 14

To produce a DNA profile the cut fragments of DNA are separated to form clear and recognisable patterns.

Question 15

What is the process of separating DNA fragments

Answer 15

It is done though electrophoresis.
The gel is then immersed in an alkali to separate the double helix strands into single strands.
The single-stranded DNA fragments are then transferred onto a membrane by Southern Blotting.

Question 16

What is the process of hybridisation

Answer 16

Radioactive or fluorescent DNA probes are added in excess to the DNA fragments on the membrane.

The DNA probes identify the micro satellite regions that are more varied that the larger mini satellite regions, The excess probes are then washed off.

Question 17

What are DNA probes

Answer 17

They are short DNA or RRNA sequences complementary to a known DNA sequence.
This binds to the complementary strands of DNA under particular conditions of PH and temperature.

Question 18

What is the process of visualisation

Answer 18

Radioactive labels are added to the DNA probes, so x-ray images are taken of the paper/membrane

If fluorescent labels are added to the DNA probes, the paper/membrane is placed under UV light and the fluorescent tags glow.

The fragments give a pattern of bars - DNA profile - which is unique to every individual

Question 19

What is the process of gel electrophoresis (1)

Answer 19

DNA fragments are put into wells in agarose gel strips, these also contain a buffering solution to maintain a constant PH.

In one of more wells, DNA fragments of a known light are used to provide a reference for fragment size.

When an electric current is passed through the electrophoresis plate, the DNA fragments in the wells at the cathode end move towards the anode at the other end.

Question 20

What is the process of gel electrophoresis (2)

Answer 20

Due to the negatively charged phosphate groups, the rate of movement depends on the mass or length of the DNA fragments the gel has a mesh like structure restricting the movement of molecules.

Smaller fragments move through the mesh more easily than the larger fragments.

When the smallest fragment reaches the anode the electric current is switched off.

Question 21

What is the process of gel electrophoresis (3)

Answer 21

The gel is then placed in an alkaline buffer solution to denature the DNA fragments., The two DNA strands of each fragment separate, exposing the bases.

These strands are transferred to nitrocellulose paper or a nylon membrane which is placed over the gel.

Question 22

What is the process of gel electrophoresis (4)

Answer 22

The membrane is covered with sheets of dry absorbent paper. Drawing the alkaline solution containing the DNA through the membrane by capillary action

Single stranded fragments of DNA are transferred to the membrane, as they are unable to pass through it.

They are transferred in the same positions as they are on the gel

Question 23

How are the DNA fragments fixed in place to the membrane

Answer 23

By using UV light or heating at 80*C

Question 24

What is DNA profiling used for

Answer 24

To solve crime

Question 25

What is a feature of crime

Answer 25

There are ony very small amounts of DNA available.

Question 26

What is PCR

Answer 26

It is a version of the natural process by which DNA is replicated allowing scientists to produce a lot of DNA from a very small sample.

Question 27

What is required for PCR to take place

Answer 27

An excess of the 4 nucleotides A,T,C,G, small primer DNA sequences, and the enzyme DNA polymerase are mixed in a vial that is placed in a thermal cycler.

Question 28

What occurs to the temperature within the PCR

Answer 28

It is controlled and changes rapidly at programmed intervals, triggering different stages of the process.

Question 29

What is a feature of how many times the reaction can occur in the machine

Answer 29

It can be repeated many times, cycling through the programmed temperature settings.

Question 30

What is step 1 of PCR

Answer 30

Separating the strands

Question 31

What is the process of separating the strands

Answer 31

The temperature in the PCR machine is increased to 90-95*C for 30 seconds, denaturing the DNA by breaking hydrogen bonds present between strands separating them

Question 32

What is step 2 of PCR

Answer 32

Annealing the primers

Question 33

What is the process of annealing the primers

Answer 33

The temperature is decreased to 55-60* and the primers bind to the ends of the DNA strands.

Question 34

What is the step 3 of PCR

Answer 34

Synthesis of DNA

Question 35

What is the process of synthesis of DNA (1)

Answer 35

The temperature is increased again to 72-75*C for 1 minute, this is the optimum temperature for DNA polymerase to work best.

Question 36

What is the process of synthesis of DNA (2)

Answer 36

It adds bases to the primer, building up complementary strands of DNA and produces a double-stranded DNA identical to the original sequence. TAQ polymerase is then used.

Question 37

Where is taq polymerase found

Answer 37

From thermophilic bacteria in hot springs.

Question 38

What are the uses of DNA profiling (1)

Answer 38

In forensic science, where PCR and DNA profiling is performed on traces of DNA left at the crime scene.

Question 39

Where can the DNA from a crime scene be obtained from

Answer 39

Blood, semen, saliva, hair roots and skin cells

Question 40

What is done with the DNA profile in crime investigations

Answer 40

The DNA profile is then compared to a suspects’ or compared to a criminal DNA database Identifying whether someone is guilty or innocent

Question 41

What is DNA profiling used for (2)

Answer 41

In paternity tests, and immigration cases to prove or disprove family relationships.

Question 42

What is DNA profiling used for (3)

Answer 42

Identifying the species to which an organism belongs Or to demonstrate evolutionary relationships between species.

Question 43

What is DNA profiling used for (4)

Answer 43

To identify individuals who are at risk of developing particular diseases. Certain microsatellites, or the repeating pattern they make have been associated with an increased risk/incidence of particular diseases.