6.1.3 - Manipulating genomes

Question 1

What is DNA sequencing ?

Answer 1

The process of determining the precise order of nucleotides within a DNA molecule

Question 2

What did Frederick Sanger and his team develop ?

Answer 2

Some techniques for sequencing nucleic acids from viruses and then bacteria

Question 3

What did Sanger’s technique involve ?

Answer 3

Radioactive labelling of bases and gel electrophoresis on a single gel

Question 4

What was Sanger’s technique called ?

Answer 4

Sanger Sequencing

Question 5

What did Sanger sequencing enable ?

Answer 5

Enabled him and his team to read sequences of 500-800 bases at a time

Question 6

What was established in 1990 ?

Answer 6

The HGP, human genome project

Question 7

What is the HGP ?

Answer 7

• Was an international project in which scientists from a number of countries worked to map the entire human genome
• Making the data available to everyone

Question 8

What did the early work of HGP involve ?

Answer 8

Sequencing the DNA of smaller, simpler organisms to refine and develop techniques

Question 9

What is involved in DNA sequencing ?

Answer 9

• The DNA is chopped into fragments and each fragment is sequenced.
• Involves terminator bases
• An A terminator will stop DNA synthesis at the location that an A base would be added, a C terminator where a C base would go, and so on.
• The terminator bases are also given coloured fluorescent tags - A is green, G is yellow, T is red and C is blue.

Question 10

What are terminator bases ?

Answer 10

Modified versions of the four nucleotide bases which stop DNA synthesis when they are included.

Question 11

Explain the process of DNA sequencing

Answer 11

• The DNA for sequencing is mixed with a primer, DNA polymerase, an excess of normal nucleotides and terminator bases.
• The mixture is placed in a thermal cycle that rapidly changes temperature at programmed intervals in repeated cycles - at 96°C the double-stranded DNA separates into single strands, at 50°C the primers anneal to the DNA strand.
• At 60°C DNA polymerase starts to build up new DNA strands by adding nucleotides with the complementary base to the single-strand DNA template.
• Each time a terminator base is incorporated instead of a normal nucleotide, the synthesis of DNA is terminated as no more bases can be added. As the chain-terminating bases are present in lower amounts and are added at random, this results in many DNA fragments of different lengths depending on where the chain terminating bases have been added during the process. After many cycles, all of the possible DNA chains will be produced with the reaction stopped at every base. The DNA fragments are separated according to their length by capillary sequencing, which works like gel electrophoresis in minute capillary tubes. The fluorescent markers on the terminator bases are used to identify the final base on each fragment. Lasers detect the different colours and thus the order of the sequence.
• The order of bases in the capillary tubes shows the sequence of the new, complementary strand of DNA which has been made. This is used to build up the sequence of the original DNA strand.
• The data from the sequencing process is fed into a computer that reassembles the genomes by comparing all the fragments and finding the areas of overlap between them. Once a genome is assembled, scientists want to identify the genes or parts of the genome that code for specific characteristics.

Question 12

What are some advancements that have taken place in DNA sequencing ?

Answer 12

• Instead of using a gel or capillaries, the sequencing reaction takes place on a plastic slide known as a flow cell.
• Millions of fragments of DNA are attached to the slide and replicated in situ using PCR to form clusters of identical DNA fragments.
• Sequencing process still uses the principle of adding a coloured terminator base to stop the reaction so an image can be taken.

Question 13

Why is DNA sequencing sometimes called next-generation sequencing ?

Answer 13

As all of the clusters are being sequenced and imaged at the same time

Question 14

What is PCR ?

Answer 14

• Polymerase Chain Reaction
• It is a version of the natural process by which DNA is replicated and allows scientists to produce a lot of DNA from the tiniest original sample

Question 15

What is in the vial that is placed in the PCR machine ?

Answer 15

• The DNA sample that is going to be amplified
• An excess of the four nucleotides
• Small primer DNA sequences
• DNA polymerase (the one that works at high temperatures)

Question 16

What are the steps in PCR ?

Answer 16

• Separating the strands
• Annealing of the primers
• Synthesis of DNA

Question 17

What occurs in the “separating the strands” stage ?

Answer 17

• Temperature in the PCR machine is increased to 90-95 degrees
• This denatures the DNA by breaking the hydrogen bonds holding the DNA strands together so they separate

Question 18

What occurs in the “Annealing of the primers” stage ?

Answer 18

• The temperature is decreased to 55-60 degrees and the primers bind to the ends of the DNA strands
• They are needed for the replication of the strands to occur

Question 19

What occurs in the “Synthesis of DNA” stage ?

Answer 19

• Temperature is increased again to 75 degrees for at least one minute - optimum temperature for the DNA polymerase
• DNA polymerase adds bases to the primer, building up complementary strands of DNA and so producing DNA identical to the original sequence
• The enzyme Taq polymerase is used, which is obtained from thermophilic bacteria that are found in hot springs

Question 20

What is the human genome ?

Answer 20

• All of the genetic material the organism contains
• This is the DNA in the nucleus and the mitochondria combined

Question 21

What are exons ?

Answer 21

Your genes, the DNA that code for proteins

Question 22

What are introns ?

Answer 22

Large non coding regions of DNA that are removed from mRNA before it is translated

Question 23

What is satellite DNA ?

Answer 23

• Short sequences that are repeated many times
• Found within introns, telomeres, and centromeres

Question 24

What are VNTRs ?

Answer 24

• Variable number tandem repeats
• Mini satellites - a sequence of 20-50 base pairs that is repeated hundreds of times

Question 25

What are STRs ?

Answer 25

• Short tandem repeats
• Micro satellites - Smaller region of just 2-4 bases repeated only 5-15 times

Question 26

What is DNA profiling ?

Answer 26

• Producing an image of the patterns in the DNA of an individual
• It is a technique employed by scientists to assist in the identification of individuals or familial relationships

Question 27

What are the 5 main stages of producing a DNA profile ?

Answer 27

• Extracting the DNA
• Digesting the sample
• Separating the DNA fragments
• Hybridisation
• Seeing the evidence

Question 28

Explain the ‘Extracting the DNA’ stage of DNA profiling

Answer 28

• DNA must be extracted from a tissue sample
• Uses PCR, allows for the tiniest fragment of tissue to give scientists enough DNA to develop a profile

Question 29

Explain the ‘Digesting the sample’ stage of DNA profiling

Answer 29

• Strands are cut into small fragments using restriction endonucleases
• These cut DNA at specific restriction sites
• They make two cuts, one through each strand of the DNA double helix
• Basically cutting the DNA into small fragments - intact mini and micro satellite regions

Question 30

What are restriction endonucleases ?

Answer 30

• Special enzymes that cut DNA into small fragments
• They give scientists the ability to cut the DNA strands at defined points in the introns

Question 31

Explain the ‘Separating the DNA fragments’ stage of DNA profiling

Answer 31

• To separate the cut fragments into a clear and recognisable pattern, electrophoresis needs to be done
  
  • It utilises the way that charged particles move through a gel medium under the influence of an electric current
• The single stranded DNA fragments are then transferred onto a membrane

Question 32

Explain the ‘Hybridisation’ stage of DNA profiling

Answer 32

• Radioactive/fluorescent DNA probes are added in excess to the DNA fragments
• They bind to the complementary strands of DNA under particular conditions
• Excess probes are washed off

Question 33

What are DNA probes ?

Answer 33

These are short DNA or RNA sequences that are complementary to a known DNA sequence

Question 34

Explain the ‘Seeing the evidence’ stage of DNA profiling

Answer 34

• If the DNA probes were radioactive, x ray images are taken
• If the DNA probes were fluorescent, paper is placed under UV light
• The fragments are a pattern of bars, which is unique to every individual except identical siblings

Question 35

What is the best known use of DNA profiling ?

Answer 35

Forensics and criminal investigations

Question 36

Explain the process of using PCR and DNA profiling in forensics

Answer 36

• PCR and DNA profiling is performed on traces of DNA left at the crime scene.
• These DNA traces can be obtained from blood, semen, saliva, hair roots, and skin cells.
• The DNA profile is compared to that of a sample taken from a suspect, or can be identified from a criminal DNA database.

Question 37

What are some other uses of DNA profiling ?

Answer 37

• DNA profiling is also used to prove paternity of a child when it is in doubt.
• It is used in immigration cases to prove or disprove family relationships.
• Identifying the species to which an organism belongs can also now be done by DNA profiling
• It is also increasingly used to demonstrate the evolutionary relationships between
  different species.

Question 38

How can DNA profiling be used to identify individuals at risk of disease ?

Answer 38

• Certain non-coding micro satellites, or the repeating patterns they make, have been found to be associated with an increased risk/incidence of particular diseases, including various cancers and heart disease.
• These specific gene markers can be identified and observed in DNA profiles.