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Biology > 6.1.3 Manipulating Genomes > Flashcards

6.1.3 Manipulating Genomes Flashcards

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1
Q

What is DNA sequencing?

A

Working out the sequences of DNA nucleotide bases.

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2
Q

What is meant by the term genome?

A

The entire genetic material of an organism

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3
Q

Why is it useful to have this knowledge of DNA sequencing?

A
  • To look at evolutionary relationships
  • To solve crime scenes
  • To look at how phenotypes are affected by genes and the environment.
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4
Q

What regions of DNA are coding?

A

Exons

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5
Q

What regions of DNA are non-coding?

A

Introns which are removed from mRNA before it is translated into a polypeptide chain

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6
Q

What are dideoxynucleotides?

A

Modified nucleotides where 2 oxygen molecules are removed

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7
Q

What effect do dideoxynucleotides have on sequencing?

A

They stop the sanger sequencing from replicating. They lack the OH group which means phosphodiester bonds cannot be made.

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8
Q

How do you amplify the amount of DNA at the beginning?

A

By using polymerase chain reaction

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9
Q

Describe the process of polymerase chain reaction (stage 1)

A
  1. Denaturation
    - Denature means to separate the DNA strands into 2 separate strands. This involved heating the DNA sample up to temperatures of 90°
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10
Q

Describe the process of polymerase chain reaction (stage 2)

A
  1. Annealing
    - Annealing means to add. In this step, primers are added to the 2 separated strands.
    - The temperature is cooled to 55°, and this helps the primers bind to the DNA.
    - The primer is an attachment that signals the DNA polymerase where to start synthesising new DNA.
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11
Q

Describe the process of polymerase chain reaction (stage 3)

A
  1. Extension
    - New DNA is created. Polymerase molecules attach to the 2 primers on the 2 DNA strands and move alone the strand.
    - As they move along they create new ‘complementary’ DNA. Temperature goes up to around 72°.
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12
Q

Process of manual sanger sequencing?

A
  1. Add dideoxynucleotides into a test tube.
  2. Add many copies of DNA (which can be amplified using PCR) and primers and free nucleotides into the same test tube.
  3. DNA polymerase binds to a primer and joins regular nucleotides to the chain.
  4. If it encounters a dideoxynucleotide is is terminated.
  5. This gives each DNA sequence a different length and mass.
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13
Q

How does gel Electrophoresis work?

A

DNA sequences are negatively charged so move towards the negative charge (cathode). DNA is placed in wells at the Anode (positive charge). DNA moves towards the Cathode. The ones that move the furthest are the smallest chains and those that don’t are larger/have more mass.

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14
Q

How does automated sanger sequencing work?

A
  1. Each type of dideoxynucleotide is labelled with a fluorescent dye.
  2. The single-stranded DNA chains are separated according to mass using a specific type of electrophoresis which takes place inside a capillary (this is called capillary electrophoresis). This has high resolution.
  3. A laser beam is used to illuminate all of the dideoxynucleotides, and a detector then reads the colour and position of each dye feeding the information into a computer.
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15
Q

What is pyrosequencing?

A

Uses sequencing by synthesis, but not by sanger sequencing. It involves synthesising a single strand of DNA, complementary to the strand to be sequenced, one base at a time, whilst detecting light emission, which base was added at each step.

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16
Q

Process of pyrosequencing

A
  1. A piece of DNA is cut into fragments.
  2. They are made single stranded and are the template DNAs. They are immobilised on a bead.
  3. A primer is added and DNA is incubated with DNA polymerase and other enzymes.
  4. Then, one of the four specifically activated nucleotides are added one at a time.
  5. If the next nucleotide is attached, there is a reaction and light is emitted which can be detected by a camera. If two or more nucleotides are found together the more light is emitted.
  6. Any unused nucleotides are degraded before the next base is added.
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17
Q

What are tandem repeats?

A

A sequence of two or more DNA bases that is repeated many times in a head-tail manner on a chromosome.

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18
Q

What is genetic engineering?

A

A process used to produce specific patterns of DNA bands from an individuals genome

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19
Q

What do VNTRs stand for?

A

Variable number tandem repeats - which are the non-coding regions (introns) which are repeating sequences.

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20
Q

What locations of the genome are VNTRs found?

A

Centromere, introns, telomeres.

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21
Q

Stage 1 of DNA profiling

A
  1. Extracting the DNA
    - Small amount of DNA are taken, they undergo PCR which amplifies the DNA so a large amount of DNA can be used.
22
Q

Stage 2 of DNA profiling

A
  1. Digesting the sample
    - The strands of DNA are cut into smaller fragments using a special enzyme called restriction endonuclease. (These act like molecular scissors). Each restriction enzyme will cut the sequence at different parts which means each fragment is varying in size.
23
Q

Stage 3 of DNA profiling

A
  1. Separating the DNA fragments
    - Electrophoresis is used to be able to see the sequence of bases in each DNA fragment.
    - The gel is then immersed in alkaline solution in order to separate the double helix into single strands.
    - Single stranded DNA is then transferred onto a membrane by southern blotting.
24
Q

Stage 4 of DNA profiling

A
  1. Hybridisation
    - Fluorescent DNA probes are added in excess to the DNA fragments on the membrane.
    - They bind to a complementary strand of DNA under particular conditions of pH or temperature.
25
Q

What are DNA probes?

A

Short sequences of DNA or RNA complementary to a known DNA sequence.

26
Q

Stage 5 of DNA profiling

A
  1. Seeing the evidence
    - If radioactive labels were added to the DNA probes, X-ray images are taken of the membrane.
    - If fluorescent labels were added to the DNA probes, the membrane is placed under UV light so the tags glow.
27
Q

What are the main uses of DNA profiling?

A
  • Its best used in forensic science, especially in criminal investigations. For example DNA and RNA traces using PCR from small amounts of samples which are left at the scene of the crime.
  • DNA is also used to prove the paternity of a child. For example it is used in immigration cases to prove or disprove family relationships.
28
Q

What is computational biology or bioinformatics?

A

They describe all different aspects of the application of computer technology to biology.

29
Q

What is bioinformatics?

A

The development of the software and computing tools needed to organise and analyse raw data.

30
Q

What is computational biology?

A

Computational biology uses bioinformatics to build theoretical models of biological systems, which can be used to predict what will happen in different circumstances.

31
Q

What is genomics?

A

The field of genetics that applies DNA sequencing methods and computational biology to analyse the structure and function of genomes.

32
Q

Advantages of analysing the genomes of pathogens

A

Sequencing genomes of pathogens including fungi, bacteria, viruses and protectionist have because increasingly cheap and fast to conduct. This allows:
- Doctors to find out the source of an infection.
- Doctors to identify antibiotic resistant strains of bacteria, ensuring antibiotics are only used when it is necessary and they will actually work saving resources.

33
Q

Uses for identifying species

A

Using traditional methods of observation, it is difficult to keep track of organisms that have been identified.
Genome analysis provides scientists with another tool to aid in species identification.

34
Q

Uses for searching for evolutionary relationships

A

It allows for scientists to understand links between evolutionary relationships between organisms. DNA sequence of different organisms is compared - because the basic mutation rate of DNA can calculate how long ago two species diverged from a common ancestor.

35
Q

What is proteomics?

A

The study of amino acid sequencing of an organism’s entire protein complement. Scientists believe by looking at DNA sequences they can look at the amino acid sequences which will tell you which amino acids are coded for.

36
Q

What are Spliceosomes?

A

During translation, introns are removed then the exons are translated together by complex enzymes known as spliceosomes. The spliceosomes may join the same exons in a variety of ways, and as a result a single gene may produce several versions of functional mRNA, which would code for different amino acids, giving different proteins.

37
Q

What is protein modification?

A

Some proteins are modified by other proteins after they are synthesised. A protein that is coded for by a gene may remain intact or it may be shortened or lengthened to give a variety of other proteins.

38
Q

What is synthetic biology?

A

The ability to sequence the genome of organisms and understand how each sequence is translated into amino acids.

39
Q

Techniques used in synthetic biology?

A
  1. Genetic engineering
  2. Use of biological systems or parts of biological systems in industrial contexts. For example immobilised enzymes and the production of drugs from micro-organisms.
  3. The synthesis of new genes to replace faulty genes for example Cystic fibrosis.
  4. The synthesis of an entire new organism replacing the original genome with a new, functional genome.
40
Q

What is genetic engineering?

A

The manipulation of a genome.

41
Q

The basic principles of genetic engineering

A

Isolating a gene from a desirable characteristic (for example insulin) and placing it into another organism, using a suitable vector.

42
Q

What is meant by ‘transgenic’?

A

Transgenic is an organism that carries the gene from another organism.

43
Q

Whats the first stage of genetic engineering?

A

Isolate the desirable gene. This is done by using an enzyme called restriction endonuclease (which are like molecular scissors)and this cuts the desired gene from the organism. Some make clean (blunt end) cuts and others make sticky end cuts.

44
Q

What are sticky ends?

A

Regions of DNA which are exposed and do not have a complementary base pair. Sticky ends make it much easier to insert the desired gene into the DNA of another organism as it can more easily bind to complementary base sequances.

45
Q

Another way a desired gene can be made?

A

Isolate the mRNA for the desired gene and use an enzyme called reverse transcriptase to produce a single strand of complementary DNA.

46
Q

What is the second stage of genetic engineering?

A

The formation of recombinant DNA. This means the DNA isolated from the restriction endonuclease must be inserted into a vector that can carry it into the host cell.

47
Q

The third stage of genetic engineering

A

The most commonly used vector is bacterial plasmids - which are small circular molecules of DNA separate from the chromosomal DNA that can replicate independently.

48
Q

What happens to the plasmids?

A

Once a plasmid gets into a new host cell it can combine with the host cell’s DNA to form what is known as a recombinant DNA. Plasmids are particularly effective in the formation of genetically engineered bacteria used, for example to make human proteins.

49
Q

What do plasmids contain?

A

A marker gene which enables scientists to determine that the bacteria has taken up the plasmid, by growing bacteria medium containing antibiotics.

50
Q

Stage 4 - transferring the vector

A

The plasmid with the recombinant DNA must be transferred into the host cell in a process called transformation.

Biology (2 decks)

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