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Biology A-Level -James > Chapter 6.3- Manipulating Genomes > Flashcards

Chapter 6.3- Manipulating Genomes Flashcards

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1
Q

What is DNA sequencing?

A

a technique that allows genes to be isolated and read, with the precise order of nucleotides within a DNA molecule being found out

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2
Q

What was the first part of Fred Sanger DNA sequencing approach?

A

technique called interrupted PCR:

  • use a single strand of DNA as a template for four experiments in separate dishes
  • Each dish contained a solution with the four bases-A,T,C and G- plus an enzyme, DNA polymerase.
  • to each dish, a modified version of one of the nucleotide bases was added
  • once a modified base was added and was incorporated into the synthesised complementary strand of DNA, no more bases could be added.
  • Each modified base was also labelled with a radioactive isotope.
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3
Q

What was the second part of Fred Sanger’s DNA sequencing approach?

A
  • following interrupted PCR, thousands of DNA fragments of varying lengths are produced as a result of the different modified bases being added to each dish
  • the fragments are then separated using a method called electrophoresis.
  • smaller fragments travelled further, so the fragments became sorted by length.
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4
Q

How was the DNA sequence determined after electrophoresis?

A

The nucleotide base at the end of each fragment was read according to its radioactive label.

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5
Q

How is DNA cloned?

A
  • gene to be sequenced is isolated, using restriction enzymes from a bacterium
  • The DNA is then inserted into a bacterial plasmid (vector)
  • the DNA is then taken to a Escherichia coli bacterium host that, when culture, divided many times, enabling the plasmid with the DNA insert to be copied many times
  • the lengths of DNA within the gene were isolated using plasmid preparation techniques and were then sequenced.
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6
Q

What was the first automated DNA sequencing machine able to do in 1986?

A
  • Fluorescent dyes instead of radioactivity were used to label the terminal bases.
  • the dyes glowed when scanned with a laser beam, and the light signature was identified by computer
  • this method dispensed with the need for technicians to read autoradiograms (used in Fred Sanger’s method)
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7
Q

What is an example of a high throughput sequencing method where sequencing genomes is fast and cheap?

A

Pyrosequencing

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8
Q

What is pyrosequencing?

A

synthesising a single strand of DNA,complementary to the strand to be sequenced, one base at a time, whilst detecting, by light emission, which bases was added as each step

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9
Q

What is the first and second stage of pyrosequencing?

A
  1. a long length of DNA is cut into fragments 300-800 base pairs long, and this is done using a nebuliser.
  2. the fragments of DNA are degraded into single-stranded DNA (ssDNA). Template DNAs which are immobilised.
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10
Q

What is the third and fourth stage of pyrosequencing?

A
  1. As sequencing primer is added to the single strand of DNA, and then incubated with DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5’ phosposulfate (APS), luciferin and one of the activated nucleotides ATP,CTP,GTP,TTP.
  2. One activated nucleotide (a nucleotide with 2 extras phosphoryl groups), such as TTP (thymine triphosphate, is incorporated into a complementary strand of DNA using the strand to be sequenced as a template. When this happens, the two extras phosphoryls are released as pyrophosphate (PPi).
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11
Q

What happens when the PPi (pyrophosphate) is generated in pyrosequencing?

A
  • PPi is converted by ATP sulphurase into ATP

- in the presence of this ATP, the enzyme luciferase converts luciferin to oxyluciferin.

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12
Q

Why does oxyluciferin allow DNA sequence to be determined?

A

The conversion of ATP to oxyluciferin generates visible light which can be detected by a camera. The amount of light generated is proportional to the amount of ATP available, indicating how many of the same type of activated nucleotide were incorporated adjacently into the complementary strand.

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13
Q

What degrades the unincorporated activated nucleotides?

A

apyrase

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14
Q

How many bases can be sequences in 10 hours?

A

400 million bases are sequenced

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15
Q

What is bioinformatics?

A

the branch of biology that deals with the large amounts of data generated by such techniques.

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16
Q

What was the Human Genome Project?

A
  • project using DNA sequencing to determine the sequence of nucleotide base pairs that make up human DNA
  • project was started in 1990 and took 15 years finishing in 2003
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17
Q

How many genes were found in the human genome?

A

Not as much as they thought- only about 24,000 genes

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18
Q

How is DNA sequencing used to make comparisons between species?

A
  • Most of the genes from the Human Genome Project are shared within other organisms, such as chimpanzees (99%) which verifies that genes that work well tend to be conserved by evolution.
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19
Q

What is the gene called that gives humans the ability to speak?

A

FOXP2

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20
Q

How can DNA sequencing be used to study evolutionary relationships?

A

Comparing genomes of closely related species confirms they evolutionary relationship, or has had the opposite effect and led to some organisms being reclassified.

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21
Q

How much of Human DNA is not shared by other humans?

A

about 0.1%

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22
Q

What are the places where substitution occurs on DNA inside humans which leads to genetic variation?

A

single nucleotide polymorphism (SNPs ‘snips’)

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23
Q

What is epigenetic?

A

study of how methylation of DNA causes changes in the expression of some genes, leads to some people getting certain diseases and some not to

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24
Q

What is methylation?

A

methylation plays a major role in regulating gene expression in eukaryotic cells. The is then mapped to help understand about diseases.

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25
Q

How has DNA sequencing allowed for the amino acids sequences of proteins to be predicted?

A

If the organisms genome has been sequenced then the primary structure of an amino acid can be determined as we know which base triplets code for which amino acid.

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26
Q

What is synthetic biology?

A

the design and building of useful biological devices and systems. it encompasses biotechnology, evolutionary biology, molecular biology, systems biology and biophysics.

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27
Q

What are two examples of applications of synthetic biology?

A
  • nanotechnology: materials can be produced for nanotechnology-e.g. amyloid fibres for making biofilms-for functions such as adhesion.
  • Novel proteins: designed proteins have been produced, for example one that is similar to haemoglobin and binds to oxygen, but not to carbon monoxide.
28
Q

What is DNA profiling?

A

refers to the practice of DNA analysis that confirms the identity of an individual.

29
Q

What are the stages in DNA profiling?

A
  • DNA is obtained from an individual- either by a mouth swab, from saliva on a toothbrush etc
  • This DNA is then digested with restriction enzymes. These enzymes cut the DNA at specific recognition sites. They will cut it into fragments, which will vary in size from individual to individual.
  • The fragments are then separated by gel electrophoresis and stained.
  • A badning pattern can be seen
  • The DNA to which the individuals is being compared to is treated with the same restriction enzymes and also subjected to electrophoresis.
  • The banding patterns of the DNA samples can then be compared.
30
Q

What are tandem repeats?

A

-repetitive segments of DNA that do not code for proteins. They may be between 10 and 100 base pairs long and they all feature the same core sequence, GGGCAGGAXG, where X can be any one of the four nucleotide bases.

31
Q

What are tandem repeats?

A
  • repetitive segments of DNA that do not code for proteins. They may be between 10 and 100 base pairs long and they all feature the same core sequence, GGGCAGGAXG, where X can be any one of the four nucleotide bases.
  • Tandem repeats occur at more than 1000 locations in the genome, and , in each of these place, they may be repeated a random number of times.
32
Q

What do tandem repeats tell us?

A
  • number of tandem repeats showed a family resemblance

- the DNA profile for each family member was unique

33
Q

In DNA profilling, what type of tandem repeats are used?

A
  • short tandem repeats (STR)

- vary from person to person and STR sequences are separated by electrophoresis.

34
Q

How is DNA profiling used in forensic science?

A

-brought about convictions of criminals, and established the innocence of many suspects and of previously wrongly convicted.

35
Q

How does DNA profiling help in maternity and paternity disputes?

A
  • half of the short tandem repeat (STR) fragments come from the mother and half from the father.
  • comparing the DNA profiles of mother, father and child can therefore establish maternity and /or paternity.
36
Q

How does DNA profiling help in the analysis of disease?

A
  • protein electrophoresis can detect the type of haemoglobin present and aid diagnosis of sickle cell anaemia.
  • varying number of repeat sequences for a condition such as Huntington disease can be detected by electrophoresis.
37
Q

What is a polymerase chain reaction (PCR)?

A

a biomedical technology in molecular biology that can amplify a shot length of DNA to thousands of millions of copies.

38
Q

What does PCR rely on to artificially replicate DNA?

A
  • DNA is made of two anti parallel backbone strands
  • each strand of DNA has a 5’ end and a 3’ end
  • DNA grows only from the 3’ end
  • base pairs pair up according to complementary base pairing rules, A with T and G with C.
39
Q

How is PCR different from DNA replication?

A
  • only short sequences( up to 10,000 base pairs, of DNA can be replicated, not entire chromosomes
  • it requires the addition of primer molecules to make the process start
  • a cycle of heating and cooling is needed to separate the DNA strands, bind primers to the strands and for the DNA strands to be replicated.
40
Q

What are the first four steps in PCR?

A
  1. The sample of DNA is mixed with DNA nucleotides, primers, magnesium ions and the enzyme Taq DNA polymerase
  2. The mixture is heated to around 94-96 degrees to break the hydrogen bonds between complimentary nucleotide base pairs and thus denature the double-stranded DNA into two single strands of DNA
  3. Mixture is cooled, so that the primers can anneal (bind by hydrogen bonding) to one end of the each single strand of DNA. This gives a small section of double-stranded DNA at the end of each single-stranded molecule.
  4. The Taq DNA polymerase enzyme molecule can now bind to the end where there is a double-stranded DNA. Taq polymerase is obtained from a bacterium that lives at high temperatures; 72 degrees is the optimum temperature for this enzyme.
41
Q

What are the last four steps in PCR?

A
  1. The temperature is raised to 72 degrees, which keeps the DNA as single strands
  2. The Taq DNA polymerase catalyses the addition of DNA nucleotides to the single-stranded DNA molecules, starting at the end with the primer and proceeding in the 5’ to 3’ direction
  3. When the Taq DNA polymerase reaches the other end of the DNA molecule, then a new double strand of DNA has been generated
  4. The whole process begins again and is repeated for many cycles.
42
Q

In the PCR process how much does the amount of DNA which is replicated increase by?

A

Increases exponentially: 1-2-4-8-16-32-64-128-so on

43
Q

what are some of the ways that PCR is applied in DNA analysis?

A
  • forensic science: small quantities of DNA can be amplified for DNA profiling , to identify criminals or to ascertain parentage.
  • detecting mutations
  • tissue typing: donor and recipient tissues can be typed prior to transplantation to reduce the risk of rejection of the transplant.
44
Q

What is electrophoresis?

A

process used to separate proteins or DNA fragments of different sizes.

45
Q

Outline briefly, how electrophoresis is carried out?

A
  • DNA samples are digested with restriction enzymes to cut them, at specific recognition sites, into fragments.
  • DNA is placed into wells of the gel
  • Gel is immersed in tank of buffer solution
  • Phosphate groups make DNA negatively charged, so DNA diffuses through the gel towards the positive electrode
  • Shorter lengths of DNA move faster than longer lengths, so move further
  • Position of fragments can be shown by a dye that stains DNA molecules
46
Q

What is a cathode?

A

the negatively charged electrode by which electrons enter an electrical device

47
Q

What is an anode?

A

the positively charged electrode by which the electrons leave an electrical device

48
Q

Does DNA have a charge and how does this effect electrophoresis?

A
  • DNA has an overall negative charge, due to its many phosphate groups
  • this results in the DNA fragments migrating towards the anode (positive electrode).
49
Q

Why is agarose gel used in electrophoresis instead of agar?

A

The main differences is that agarose is chemically more simple, and also has a neutral charge, and is therefore
preferred for this technique as it is less likely to interfere with the DNA structure.

50
Q

How are proteins separated by the process of electrophoresis?

A
  • similar to separating DNA fragments
  • BUT, often carried out in the presence of a charged detergent such as sodium dodecyl (SDS): this equalises the surface charge on the molecule and allows the proteins to separate as they move through the gel, according to their molecular mass.
  • this is necessary because different proteins have different surface charges
51
Q

What is a DNA probe?

A

short, single stranded pieces of DNA that is complimentary to a section of the DNA being investigated

52
Q

How does a DNA probe work?

A
  • the probe is labelled with either a radioactive (exposed by photographic film) or fluorescent marker (emits a colour to exposure to UV light)
  • Copies of the probe will anneal to complementary sequence on the section of the DNA being investigated.
53
Q

Why are DNA probes useful?

A
  • Locate a desired gene for genetic engineering
  • Identify the same gene on different genomes
  • Identify the presence or absence of an allele for a particular genetic disease
54
Q

What are microarrays?

A

This is when the DNA probes are fixed onto a surface such as agarose gel, and then the DNA under investigation is added and can reveal the presence of mutated alleles that match the fixed probes, because the sample DNA will anneal to any complimentary fixed probes.

55
Q

What do microarrays allow us to do?

A
  • genetic screening
  • personalised medicine
  • genetic counselling
56
Q

What does genetic engineering mean( recombinant DNA)?

A

A composite DNA molecule created in vitro by joining foreign DNA with a vector molecule such as a plasmid.

57
Q

What are the four stages of genetic engineering?

A
  1. The required gene is obtained
  2. A copy of the gene is placed inside a vector
  3. The vector carries the gene into a recipient cell
  4. The recipient cell expresses the novel gene
58
Q

What are the ways that the required gene in genetic engineering can be obtained?

A
  1. mRNA from the gene being expressed can be obtained, and then the enzyme reverse transcriptase can catalyse the formation of a single strand of complementary DNA (cDNA) using the mRNA as a template. The mRNA is then digested which creates single-stranded DNA (ssDNA).The addition of primers and DNA polymerase can make this ssDNA into a double-stranded length of DNA, whose base sequence codes for the original protein.
  2. The gene can be synthesised using a automated polynucleotide synthesiser if the sequence of the gene is known
  3. If the scientists know the sequence of that the gene, they can design polymerase chain reaction (PCR) primers to amplify the gene from the genomic DNA.
  4. A DNA probe can be used to locate a gene within the genome and the gene can then be cut out using restriction enzymes
59
Q

What are the different ways that a gene can be placed inside a vector?

A
  • Plasmids can be obtained from organisms and mixed with restriction enzymes (restriction endonuclease) that will cut the plasmid at specific recognition sites
  • the cut plasmid ten exposes unpaired nucleotide bases, called sticky ends
  • if free nucleotide bases, complementary to the sticky ends of the plasmid, are added to the ends of the gene to be inserted, then the gene and cut plasmid should anneal(bind). DNA ligase enzymes catalyses the annealing. This forms a recombinant DNA/plasmid
  • A gene may be sealed into an attenuated (weakened) virus that could carry it into a host cell
60
Q

What is a plasmid?

A

small loops of DNA in prokaryotic cells

61
Q

What is a restriction enzyme?

A

endonuclease enzymes that cleave DNA molecules at specific recognition sites

62
Q

What is a vector?

A

in gene technology, anything that can carry/insert DNA into a host organism; examples of such vectors include plasmids, viruses and certain bacteria

63
Q

What are the ways that the vector can enter the recipient cell in genetic engineering?

A
  • Heat shock treatment: if bacteria are subjected to alternating periods of cold 0 degrees and heat 42 degrees in the presence of calcium chloride, their walls and membranes will become more porous and allow in the recombinant vector. This is because the positive calcium ions surround the negatively charged parts of both the DNA molecules and phospholipids in the cell membrane, thus reducing repulsion between the foreign DNA and the host cell membranes.
  • Electroporation: a high voltage pulse is applied to the cell to disrupt the membrane and make it more porous.
  • Electrofusion: electrical fields help to introduce DNA into cells
  • Transfection-DNA can be packaged into a bacteriophage, which can then transfect the host cell
  • T1 (recombinant) plasmids are inserted into the bacterium Agrobacterium tumefaciens, which infects some plants and naturally inserts its genome into the host cell genomes.
  • ‘gene gun’: small pieces of gold or tungsten are coated with the DNA and shot into the plant cells
64
Q

How do you identify the vector that has taken up the gene in genetic engineering?

A
  • antibiotic resistance gene markers are used
  • When inserting the gene into the plasmid we make sure that it is inserted in the middle (disrupts) an antibiotic resistance gene
  • this is done by making sure that the restriction endonuclease that it used only cuts the site that is in the middle of the antibiotic resistance gene (tetracycline resistance gene).
  • also make sure that the plasmid has another resistance gene (ampicillin resistance gene) that codes for the resistance of a different antibiotic.
  • the plasmids are then moved onto agar plate and allowed to grow. They are then transferred to a plate which contains ampicillin. Here all the colonies grow because both the recombinant and un-recombinant genes have the ampicillin-resistance gene
  • then they are transferred to an agar plate contains tetracycline agar-only non-recombinant colonies grow on this plate.
  • the plasmids which are recombinant are found on the ampicillin plate and these should be then incubated and grown.
65
Q

Why is using transgenenic bacteria dangerous?

A

they have resistance to some antibiotics and if they were to escape from the laboratory into the wild than this could have some negative effects.

66
Q

What are some potential hazards associated with genetic modification (GM)?

A

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Biology A-Level -James (18 decks)

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