chapter 21

Question 1

genome of an organism

Answer 1

all of the genetic material it contains - for eukaryotes including ourselves, that is the DNA in the nucleus and the mitochondria combined.

Question 2

chromosomes are made up of…

Answer 2

hundreds of millions of DNA base pairs, but your genes, the 20-25 000 regions of the DNA that code for proteins, only make up about 2% of your total DNA - They are called exons.

Question 3

introns

Answer 3

The large non-coding regions of DNA that are removed from messenger (m)RNA before it is translated into a polypeptide chain

Question 4

satellite DNA

Answer 4

found within introns, telomeres, and centromeres
short sequences of DNA that are repeated many times

Question 5

minisatellite

Answer 5

a sequence of 20-50 base pairs will be repeated from 50 to several hundred times
These occur at more than 1000 locations in the human genome and are also known as variable number tandem repeats (VNTRs).

Question 6

microsatellite

Answer 6

• a smaller region of just 2-4 bases repeated only 5-15 times - They are also known as short tandem repeats (STRs)
• These satellites always appear in the same positions on the chromosomes, but the number of repeats of each mini- or microsatellite varies between individuals, as different lengths of repeats are inherited from both parents.
• So just as in the coding DNA, only identical twins will have an identical satellite pattern, although the more closely related you are to someone, the more likely you are to have similar patterns.
• These patterns in the non-coding DNA were discovered by Professor Sir Alec Jeffreys and his team at Leicester University in 1984.

Question 7

DNA profiling

Answer 7

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

Question 8

five main stages in producing a DNA profile:

Answer 8

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

Question 9

step 1 of dna profiling
- Extracting the DNA

Answer 9

The DNA must be extracted from a tissue sample.

When DNA profiling was first discovered, relatively large samples were needed - about 1 ug of DNA, equivalent to the DNA from the nuclei of about 10000 human cells.

Now, using a technique called the polymerase chain reaction (PCR), the tiniest fragment of tissue can give scientists enough DNA to develop a profile.

Question 10

step 2 of dna profiling - Digesting the sample

Answer 10

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

Different restriction endonucleases cut DNA at a specific nucleotide sequence, known as a restriction site or recognition site.

All restriction endonucleases make two cuts, once through each strand of the DNA double helix.

There are many different restriction endonucleases - the recognition sequences and cut sites of three examples are given in Table 1.

Restriction endonucleases give scientists the ability to cut the DNA strands at defined points in the introns.

They use a mixture of restriction enzymes that leave the repeating units or satellites intact, so the fragments at the end of the process include a mixture of intact mini- and microsatellite regions.

Question 11

the recognition sequences and cut sites of three examples are given in Table 1:

Answer 11

Question 12

step 3 of dna profiling - Separating the DNA fragments

Answer 12

To produce a DNA profile, the cut fragments of DNA need to be separated to form a clear and recognisable pattern.

This is done using electrophoresis, a technique that utilises the way charged particles move through a gel medium under the influence of an electric current.

The gel is then immersed in alkali in order to separate the DNA double strands into single strands.

The single-stranded DNA fragments are then transferred onto a membrane by Southern blotting.

Question 13

step 4 of dna profiling -Hybridisation

Answer 13

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

DNA probes are short DNA or RNA sequences complementary to a known DNA sequence.

They bind to the complementary strands of DNA under particular conditions of pH and temperature.

This is called hybridisation.

DNA probes identify the microsatellite regions that are more varied than the larger minisatellite regions.

The excess probes are washed off.

Question 14

step 5 of dna profiling - Seeing the evidence

Answer 14

If radioactive labels were added to the DNA probes, X-ray images are taken of the paper/membrane.

If fluorescent labels were added to the DNA probes, the paper/membrane is placed under UV light so the fluorescent tags glow.
This is the method most commonly used today.

The fragments give a pattern of bars - the DNA profile - which is unique to every individual except identical siblings.

Question 15

Gel Electrophoresis Setup:

Answer 15

DNA fragments are put into wells in agarose gel strips, which also contain a buffering solution to maintain a constant pH.
In one or more wells (usually the first and last), DNA fragments of known length are used to provide a reference for fragment sizing.

Question 16

Electrophoresis Process:

Answer 16

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

This is due to the negatively charged phosphate groups in the DNA fragments.

The rate of movement depends on the mass or length of the DNA fragments - the gel has a mesh-like structure that resists the movement of molecules.

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

Therefore, over a period of time, the smaller fragments move further than the larger fragments.

Question 17

Post-Electrophoresis Steps:

Answer 17

When the faster smallest fragments reach the anode end of the gel, the electric current is switched off.

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.

In a technique called Southern blotting (named after its inventor, Edwin Southern), these strands are transferred to a nitrocellulose paper or a nylon membrane, which is placed over the gel.

The membrane is covered with several sheets of dry absorbent paper, drawing the alkaline solution containing the DNA through the membrane by capillary action (Figure 6).

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

They are transferred in precisely the same relative positions as they had on the gel.

They are then fixed in place using UV light or heated at 80°C.

Question 18

gel Electrophoresis set up diagram

Answer 18

Question 19

Polymerase chain reaction (PCR):

Answer 19

DNA profiling is often used in solving crimes and only very tiny amounts of DNA may be available.

The PCR 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.

The DNA sample to be amplified, an excess of the four nucleotide bases A, T, C, and G (in the form of deoxynucleoside triphosphates), small primer DNA sequences, and the enzyme DNA polymerase are mixed in a vial that is placed in a PCR machine (also called a thermal cycler).

The temperature within the PCR machine is carefully controlled and changes rapidly at programmed intervals, triggering different stages of the process

The reaction can be repeated many times by the PCR machine, which cycles through the programmed temperature settings.

About 30 repeats gives around one billion copies of the original DNA sample - more than enough to carry out DNA profiling.

Question 20

three steps in the polymerase chain reaction

Answer 20

Step 1. Separating the strands:

Step 2. Annealing of the primers:

Step 3. Synthesis of DNA:

Question 21

Step 1 of PCR. Separating the strands:

Answer 21

The temperature in the PCR machine is increased to 90-95°C for 30 seconds, this denatures the DNA by breaking the hydrogen bonds holding the DNA strands together so they separate.

Question 22

Step 2 of PCR. Annealing of the primers:

Answer 22

The temperature is decreased to 55-60°C and the primers bind (anneal) to the ends of the DNA strands.
They are needed for the replication of the strands to occur.

Question 23

Step 3 of PCR. Synthesis of DNA:

Answer 23

The temperature is increased again to 72-75°C for at least 1 minute, this is the optimum temperature for DNA polymerase to work best.
DNA polymerase adds bases to the primer, building up complementary strands of DNA and so producing double-stranded DNA identical to the original sequence.
The enzyme Taq polymerase is used, which is obtained from thermophilic bacteria found in hot springs.

Question 24

diagram of the main steps in PCR:

Answer 24

Question 25

The uses of DNA profiling:

Answer 25

Forensic Applications:
Non-Forensic Identification:
Medical and Health Applications:

Question 26

Forensic Applications:

Answer 26

Its best known use is in the field of forensic science, especially criminal investigations as 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.
DNA profiling is an extremely useful tool in providing evidence for either the guilt or innocence of a suspect.

Question 27

Non-Forensic Identification:

Answer 27

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, which is much more accurate than any of the older methods.
It is also increasingly used to demonstrate the evolutionary relationships between different species.

Question 28

Medical and Health Applications:

Answer 28

Another valuable use of DNA profiling is in identifying individuals who are at risk of developing particular diseases.
Certain non-coding microsatellites, 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.
The information that scientists can obtain from DNA profiling is often used together with the more detailed information obtained from DNA sequencing (Topic 21.2) to make more confident risk assessments for different diseases.

Question 29

Pitfalls of profiling:

Answer 29

One of the earliest recorded cases of mistaken DNA identity occurred in the UK in 2000.
Raymond Easton was in the advanced stages of Parkinson’s disease - he could hardly dress himself yet he was arrested and charged with a burglary that happened over 200 miles from his home.
The arrest was based solely on DNA evidence.
Four years earlier, Raymond had been involved in a family dispute that had got out of hand.
He received a police caution and his DNA was taken and kept on file. DNA from the 2000 burglary scene appeared to match Raymond’s profile.
Raymond protested his innocence and had a strong alibi for the time of the burglary, so eventually a more rigorous DNA test, looking at satellites in 10 loci rather than the original six, was carried out.
None of the additional satellites matched Raymond’s DNA and so the charges were dropped.
This misidentification had been caused by an extremely improbable, but not impossible, coincidental DNA profile match

Question 30

DNA sequencing

Answer 30

the process of determining the precise order of nucleotides within a DNA molecule.
This knowledge is invaluable in various scientific applications, from diagnostics to biotechnology.

Question 31

The beginning of DNA sequencing:
Early DNA Sequencing Techniques:1

Answer 31

DNA sequencing was just an aspiration for scientists until Frederick Sanger and his team developed some techniques for sequencing nucleic acids from viruses and then bacteria.
The technique involved radioactive labelling of bases and gel electrophoresis on a single gel.

Question 32

Sanger Sequencing Breakthrough: 2

Answer 32

The processes were carried out manually, so it took a long time, but eventually, in the 1970s, the technique now known as Sanger sequencing enabled Sanger and his team to read sequences of 500-800 bases at a time.

The first entire genome that they sequenced was just over 5000 bases long and belonged to phiX174, a virus that attacks bacteria.

They went on to sequence many other genomes, including the 16000 base pairs of human mitochondrial DNA.

In 1980, Frederick Sanger was awarded the Nobel Prize for his work on sequencing DNA - this was his second Nobel Prize, his first was in 1958 for determining the sequence of the amino acids in insulin.

Question 33

Automation and Capillary Sequencing: 3

Answer 33

These DNA sequencing techniques are continually being refined.

One such development was the swapping of radioactive labels for coloured fluorescent tags, which led to scaling up and automation of the process.

This in turn led to the capillary sequencing version of the Sanger sequencing method that was used during the Human Genome Project (HGP), and similar techniques that are used today.

Question 34

The human genome project part 1

Answer 34

In 1990, the HGP was established.
It was a massive international project in which scientists from a number of countries worked to map the entire human genome, making the data freely available to scientists all over the world.

The early work involved sequencing the DNA of smaller, simpler organisms to refine and develop the techniques.

In 1995, after 18 months of work, scientists completed the 1.8 million base pair genome of the bacterium Haemophilus influenza.

Question 35

The human genome project part 2

Answer 35

By 1998, the UK team at the Sanger Centre and a US team at Washington University had sequenced the genome of Caenorhabditis elegans (C. elegans), a nematode worm widely used in scientific experiments, before applying the technique to the three billion base pairs of the human genome itself.

The aim was to complete the HGP in 15 years but the automation of sequencing techniques and the development of more powerful, faster computers meant that the first draft of the human genome was ready in 2000, and the first complete human genome sequence was published in 2003, two years ahead of schedule and under budget.

Question 36

Principles of DNA sequencing:

Answer 36

The DNA is chopped into fragments and each fragment is sequenced.

The process involves terminator bases, modified versions of the four nucleotide bases, adenine (A), thymine (T), cytosine (C), and guanine (G), which stop DNA synthesis when they are included.

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.

The description of the sequencing process (capillary method) explained here is a simplified version of a technique, has largely been overtaken by much more complex methods - but the basic principles remain the same

Question 37

5 Basic principles/steps of DNA sequencing:

Answer 37

1. The DNA for sequencing is mixed with a primer, DNA polymerase, an excess of normal nucleotides (containing bases A, T, C, and G) and terminator bases.
2. The mixture is placed in a thermal cycler
3. At 60°C DNA polymerase starts to build up new DNA strands
4. 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.
5. The order of bases in the capillary tubes shows the sequence of the new, complementary strand of DNA which has been made.

Question 38

Step 1 of DNA sequencing

Answer 38

The DNA for sequencing is mixed with a primer, DNA polymerase, an excess of normal nucleotides (containing bases A, T, C, and G) and terminator bases.

Question 39

step 2 of DNA sequencing

Answer 39

The mixture is placed in a thermal cycler - a piece of equipment as used for PCR (Topic 21.1, DNA profiling) 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.

Question 40

step 3 of DNA sequencing

Answer 40

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.

Question 41

step 4 of DNA sequencing

Answer 41

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.

Question 42

step 5 of DNA sequencing

Answer 42

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.

Medical researchers want to identify regions that are linked with particular diseases.

Many genomes are freely available online - anyone who chooses to can have a look at them

Question 43

Next-generation sequencing:
early DNA Sequencing Challenges:

Answer 43

Early on, working out the base sequence of even short strands of DNA was difficult and time-consuming for scientists using the original Sanger sequencing method.

Question 44

Next-generation sequencing:
Evolution of Sequencing Technologies:

Answer 44

DNA sequencing technologies have become faster and more automated as they have been developed.

Recently, technological developments have led to new, automated, high-throughput sequencing processes.
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.

The sequencing process still uses the principle of adding a coloured terminator base to stop the reaction so an image can be taken.

As all of the clusters are being sequenced and imaged at the same time, the technique is known as ‘massively parallel sequencing’ and sometimes referred to as ‘next-generation sequencing

Question 45

Massively Parallel Sequencing and Applications:

Answer 45

The process of massively parallel sequencing is integrated with state-of-the-art computer technology and is constantly being refined and developed.
These high-throughput methods are extremely efficient and very fast - the 3 billion base pairs of the human genome can be sequenced in days and those of a bacterium in less than 24 hours.
High-throughput sequencing also means that the cost has fallen, so more genomes can be sequenced.
These techniques are being used by projects such as the 100,000 Genomes Project.
They open up the range of questions that scientists can ask and enable us to use the information from the genome in many new and different ways (21.3, Using DNA sequencing).