Manipulating genomes

Question 1

What is a gene?

Answer 1

A section of DNA that codes for a protein (polypeptide) which results in a characteristic.

Question 2

What is a locus?

Answer 2

The position on a chromosome where a particular allele is found.

Question 3

What is a genome?

Answer 3

All of the genetic material an organisms contains.

Question 4

What were/are the aims of the human genome project?

Answer 4

To work out the order of sequences of all the 3 billion base pairs in the human genome.
To identify all the genes
To develop faster and cheaper methods for sequencing DNA

Question 5

What is the 1st step in producing a DNA profile?

Answer 5

DNA extraction - DNA is extracted from the tissue sample

Question 6

What is the 2nd step in producing a DNA profile?

Answer 6

Digestion - Fragment is cut into smaller pieces called satellites by restriction endonucleases. They cut through both strands of DNA. Each type of endonuclease cuts at a specific base sequence called the recognition site. If a mixture of endonuclease are used then predictable satellites will be produced.
Mini satellites are repeats of sequences which are 20-50 base pairs long and micro satellites are 2-4 base pairs long.

Question 7

What is the 3rd/4th step in producing a DNA profile?

Answer 7

Separation of fragments - fragments need to be separated and shown clearly which is done via electrophoresis. DNA has a slight negative charge and so is able to move when the gel has electricity passed through it. The gel is then immersed in alkali so the strands are broken apart. These single strands are then transferred onto a nylon membrane in excess called southern blotting.

Question 8

What is the 5th step in producing a DNA profile?

Answer 8

Hybridisation - radioactive or fluorescent DNA probes are added to the membrane. DNA probes are short sequences of DNA that are complimentary to specific sequences of DNA. They bind to DNA under specific conditions of temperature and ph. They attach to particular microsatellites (hybridisation).

Question 9

What is the 6/7th step in producing a DNA profile?

Answer 9

Development - if radioactive probes were used, an X-ray is taken off the blot. If fluorescent probes are used, then UV light is used to look at the blot. This gives a pattern of bars that can be seen that are specific to the sample taken.

Question 10

What is PCR and why is it used?

Answer 10

Amplifying DNA - amplifies small amounts of DNA - used to make identical copies of DNA. Can be used on crime scenes - artificial DNA replication. Repeated to give huge numbers. The machine used in this process is called a Thermocycler.

Question 11

What happens during step 1 of PCR?

Answer 11

Temp is increased to 95 for 30 secs - this breaks the hydrogen bonds between nitrogenous bases breaking apart the strands of DNA.
Samples, polymers, DNA bases and primers are added.
If temp is left high for too long histone proteins around DNA will be damaged.
Starts with 1 strand of DNA.

Question 12

What happens during step 2 of PCR?

Answer 12

Temp is reduced to 55 - this allows primers to bind (or anneal) to the ends of each DNA strand by complimentary base pairings - choose correct primers.

Question 13

What happens during step 3 of PCR?

Answer 13

Temp is raised to 72
DNA polymerase attaches at the primer and begins adding complimentary base pairs to the chain - free DNA nucleotides from the original mix. Known as extension - making a complimentary strand of DNA for each strand.
Specialised DNA polymerase called Taq polymerase from thermophilus bacteria is used at these higher temps - our enzymes denature at these temps.

Question 14

What is needed for PCR?

Answer 14

Excess nucleotides (A,T,C,G)
Primer DNA sequences ( short sequence of DNA that provides a starting point for DNA synthesis
The enzyme DNA polymerase
Sample of DNA

Question 15

Whats the formula for calculating numbers of DNA strands from PCR?

Answer 15

2^n
n = number of generations or cycles

Question 16

Why may the calculated value for number of DNA strands from PCR not be achieved in practice?

Answer 16

damage to template
one strand doesn’t separate properly
primers fail to attach
lack of primers/free nucleotides

Question 17

What is electrophoresis?

Answer 17

Where molecules are separated according to their size/mass and their net overall charge. Different sizes of fragments have different masses, the smaller the fragment the quicker it moves, larger molecules move slower. DNA is negatively charged so its going to move to the anode(+)

Question 18

What are the steps of electrophoresis?

Answer 18

1. DNA is cut into fragments by restriction enzymes
2. Pour agarose gel into a gel tray and leave it to solidify
3. A row of wells is created at one end of the tray by a comb
4. DNA segments loaded into wells with micropipette
5. Gel plate is immersed in charged buffer solution - DNA fragments move through gel toward positive electrode - electricity is passed through gel - cathode is near the wells.
6. Add probes to make DNA visible - radioactive proves show up under x-ray OR fluorescent show up under UV light.
7. The bands of DNA in each sample can be compared for similarities and differences.

Question 19

What happens during stages 1-2 of DNA sequencing?

Answer 19

DNA template strand chopped up, mixed with primer, bases, free DNA nucleotides, DNA polymerase and terminator bases (STOP) is placed into a thermal cycler. Each type of nucleotide has a fluorescent probe attached. Put temp up and separate DNA into 2 single strands. Turn temp down and allow the primers to anneal to the DNA strand.

Question 20

What happens during stage 3 of DNA sequencing?

Answer 20

Each time a terminator base is added a strand terminates until all possible chains produced. Largest fragments moves least distance from the +ve electrode,

Question 21

What happens during stage 4 of DNA sequencing?

Answer 21

Readout from capillary tubes: DNA fragments seperated by electrophasis in capillary tubes by mass and lasers detect the colours and the sequence.

Question 22

What happens during stage 5 of DNA sequencing?

Answer 22

Computer analysis of all data to give original DNA sequence. Order of colours tells us the order of the bases, - looks like a mass spectrometer.

Question 23

Whats a benefit of DNA sequencing this way?

Answer 23

Cheaper, quicker and more efficient.

Question 24

What is the difference between DNA profiling and DNA sequencing?

Answer 24

DNA profiling is the production of genetic fingerprint, unique to a person, based on particular sections of DNA whereas DNA sequencing is a process which determines the precise order bases in DNA sequences.

Question 25

What is massively parallel sequencing?

Answer 25

More automated
Sequencing takes place on a plastic slide known as a flow cell
Millions of fragments are attached to the cell and are replicated on the slide by PCR to form clusters of identical fragments
Still uses coloured terminator bases
All the clusters can be read at once
Sequence’s sequenced at the same time
Uses state of the art computer technology
Rapid
Lower cost
Very efficient

Question 26

What is pyrosequencing?

Answer 26

Fragments 300-800 bp long
Add to a bead and amplify using PCR
Add bead to well which contains the enzymes luciferase and sulfurylase
Add a nucleotide - if this is complimentary to the fragment DNA, the enzymes will convert the energy to a flash of light
Wash the well
No pairing = no light
Two consecutive pairs = twice the light
Computers read the light flashes

Question 27

What is nanopore sequencing?

Answer 27

Uses flow cells which contain an array of tiny holes - nanopores embedded in an electro-resistant membrane
Each nanopore corresponds to its own electrode connected to a channel and sensor chip, which measures the electric current that flows through the nanopore.
When a molecule passes through a nanopore the current is disrupted to produce a characteristic (squiggle)
The squiggle is then decoded using base calling algorithms to determine the DNA or RNA sequence in real time.

Question 28

How is bacteria used to produce human insulin?

Answer 28

1. Obtain DNA - extract from organism (human cell)
2. cDNA (copy DNA - is double stranded) - mRNA is used as its edited (introns removed) - perfect copy to make insulin. Reverse transcriptase is used.
   3.Amplify DNA - PCR used to increase amount of DNA. cDNA is put into the thermal cycler. Multiple identical copies of cDNA made.

Plasmid inserted into bacteria via 2 ways:
1. Electroporation - holes punched into membrane by electricity, plasmids taken up.
2. Electrofusion - tiny currents applied to membranes of two cells - membranes fuse.

Question 29

How are sticky ends formed?

Answer 29

Sugar-phosphate backbone of DNA is cut using restriction enzymes - breaks the hydrogen bonds between bases.
Restriction enzymes will recognise the palindromic (ATCCTA) sequence and make a cut within its recognition site on both top and bottom strands. In these cases the cuts are staggered cuts. Unpaired bases are left on each DNA strand creating sticky ends.

Question 30

How is the recombinant plasmid made?

Answer 30

DNA from cell which manufactures the desired protein (insulin)
DNA and plasmid are both cut using the same restriction endonuclease
DNA fragments and open plasmids are mixed together with DNA ligase - complimentary sticky ends are joined - hydrogen bonds are reformed.

Question 31

What are marker genes?

Answer 31

Plasmids with two antibiotic resistance genes - cut with restriction enzymes
Mix with ligase - recombinant plasmid, no plasmid, non-recombinant plasmid. OR with fluorescent if glow - plasmid taken.