Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

A Level Biology > 6.1.3 Manipulating Genomes > Flashcards

6.1.3 Manipulating Genomes Flashcards

You may prefer our related Brainscape-certified flashcards:
AP® Biology flashcards Biology 101 flashcards
1
Q

What is genetic engineering?

A
  • changing the genetic makeup of an organism
  • genes are taken from one organism and placed into another
  • to form recombinant DNA
  • the protein coded by the gene is then produced by the transgenic organism
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is recombinant DNA?

A
  • DNA from different organisms/sources joined together by complementary binding
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Why do we genetically engineer organisms?

A
  • to improve or introduce a feature in the recipient:
  • herbicide resistant gene: can kill weeds, not harm crop
  • growth controlling gene promotes muscle growth in cattle
  • engineer organisms to produce useful products:
  • bacteria: human insulin
  • golden rice: vitamin A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Give an overview of gene transfer processes

A
  1. Gene is identified and cut out or made
  2. Multiple copies of the gene are produced
  3. Gene is inserted into vector for delivery into required cell
  4. Gene inserted into recipient cells by vector and cells express the gene
  5. Genetically transformed cells identified and cloned
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Describe how to obtain the required gene in genetic engineering

A
  • mRNA can be obtained from cells where the gene is expressed
  • reverse transcriptase is used to form a single strand of complementary DNA (cDNA) using the mRNA as a template
  • addition of primers and DNA polymerase can make this cDNA into a double-stranded length of DNA with a base sequence coding for the protein

If scientists know the nucleotide sequence of the gene:

  • the gene can be synthesised using automated polynucleotide synthesis
  • or the gene can be amplified (copied) from the genomic DNA from polymerase chain reaction (PCR)
  • or a DNA probe can be used to locate a gene within the genome and then gene can be cut out using restriction enzymes
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What are restriction endonuclease enzymes?

A
  • made by bacteria and archaea to protect from phage virus attack
  • they cut up foreign viral DNA, preventing the viruses from making copies
  • the prokaryotic DNA is protected from the action of these endonucleases by being methylated at the recognition sites
  • named according to the bacterium they have been obtained from e.g. EcoR1
  • used in molecular biology and biotechnology as they recognise specific sequences within a length of DNA and cleave the molecule there
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

How do restriction endonuclease enzymes work?

A
  • cut DNA at specific recognition sites which are 4-6 base pairs long
  • enzymes recognise a palindromic sequence (e.g. from 5’ to 3’)
  • some make a staggered cut leaving sticky ends, other make a cut that produces blunt ends
  • some need Mg2+ as cofactors
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

How are multiple copies of the gene made in genetic engineering?

A
  • by using polymerase chain reaction (PCR)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How can you place the gene into a vector for delivery into recipient cell?

A

Using plasmids:

  • plasmids from bacteria can be mixed with restriction enzymes that cut the plasmid DNA at specific recognition sites
  • the cut plasmid has exposed unpaired nucleotide bases called sticky ends
  • if free nucleotide bases, which are complementary to the sticky ends of the plasmid, are added to the ends of the gene to be inserted, then the gene and plasmid should anneal (bind)
  • DNA ligase catalyses this annealing

Other vectors:

  • seal the gene into an attenuated (weakened) virus that could carry it to a host cell
  • place the gene inside liposomes
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What does DNA ligase do?

A
  • ligase links nucleotides together
  • catalyses the formation of covalent (phosphodiester bonds)
  • sealing sugar-phosphate backbone
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Why do you need various methods to aid vector getting into recipient cell?

A
  • DNA does not easily cross the recipient’s plasma membrane
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What methods help the vector to get into the recipient cell?

A
  • heat shock treatment: subject bacteria to alternating period of cold (0ºC) and heat (42ºC) in the presence of calcium chloride
  • walls and membrane will become more porous and allow in the recombinant vector
  • this is because calcium ions surround the negative part of both DNA molecules and phospholipids in the cell membrane. this reducing repulsion between foreign DNA and host cell membranes
  • electroporation: a high voltage pulse is applied to disrupt cell membranes
  • electrofusion: electrical fields can help to introduce DNA into cells
  • transfection: DNA can be packaged into a bacteriophage which can the infect the host cell
  • Ti (recombinant) plasmids are inserted into the bacterium Agrobacterium tumefaciens which infects some plants and naturally inserts its genome into host cell genomes
  • Gene gun: small pieces of gold/tungsten are coated with DNA and shot into plant cells
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Why do you have to identify cells that have been successfully taken up the gene?

A
  • not all bacteria contain the gene because
  • some bacteria may not have taken up a plasmid
  • some plasmids might have sealed back up after being cut with the restriction enzyme, so they didn’t take in the gene
  • the only bacteria we want are the ones that have the plasmid that contains the gene of interest
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

How do you identify the cells that have been successfully taken up by the gene and clone them in genetic engineering?

A
  • we use genetic markers:
  • replica plating and antibiotic resistance genes
  • fluorescent marker gene from jelly fish
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What is reverse transcriptase?

A
  • retroviruses, such as HIV, which contain RNA that inject into the host genome, have reverse transcriptase enzymes that catalyses the production of cDNA
  • they use their RNA as a template
  • this is the reverse of transcription
  • these enzymes are useful for genetic engineering
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

How is insulin made from GM bacteria?

A
  • scientists can obtain mRNA from beta cells of islet of Langerhans in the human pancreas
    1. adding reverse transcriptase enzymes makes a single strand of cDNA and treatment with DNA polymerase makes a double strand: the gene
    2. addition of free unpaired nucleotides at the ends of the DNA produces sticky ends
    3. with ligase enzyme, the insulin gene can be inserted into plasmids extracted from E.coli bacteria
  • these are now called recombinant plasmids, as they contain inserted DNA
    4. E.coli bacteria are mixed with recombinant plasmids and subjected to heat shock in the presence of calcium chloride ions, so they will take up the plasmids
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Why does bacteria have to be safely contained when making insulin?

A
  • transgenic bacteria have resistance to some antibiotics, we do not want them to escape from labs into the wild
  • therefore, they have a gene knocked out, which means they cannot synthesise a particular nutrient
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

How did Fred Sanger sequence DNA in 1975?

A
  • he used a single strand of DNA as a template for four experiments
  • each dish contained a solution with the four bases plus DNA polymerase
  • a modified version of one of the DNA bases was added to each dish
  • it was modified in the way that once incorporated into the synthesised complementary strand of DNA, no more bases could be added
  • each modified base was also labelled with a radioactive isotope
  • as the reaction progressed, thousands of DNA fragments of varying lengths were generated
  • DNA fragments were passed through a gel by electrophoresis
  • smaller fragments travelled further, so they were sorted by length
  • the nucleotide base at the end of each fragment was read according to its radioactive label
  • although this was efficient and safe, he had to count each base one by one, so it was time-consuming
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

How was DNA cloned to allow them to be sequenced in early DNA sequencing?

A
  • gene was isolated using restriction enzymes from a bacterium
  • DNA is then inserted into a bacterial plasmid and then into an E.coli host, which divided many times, enabling the plasmid with the DNA to be copied
  • these lengths of DNA were then isolated using plasmid preparation techniques and then sequenced
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

When was the first DNA sequencing machine developed?

A
  • in 1986 by California(IT)
  • used fluorescent dyes instead of radioactive
  • technicians had to read autoradiograms
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

What is high throughput sequencing?

A
  • a variety of approaches was used to develop fast, cheap methods to sequence genomes
  • e.g. pyrosequencing
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

Describe pyrosequencing incomplete…

A
  • it involves synthesising a single strand of DNA, complementary to the strand to be sequenced, one base at a time, while detecting, by light emission, which base was added at each step
    1. a long length of DNA to be sequenced is mechanically cut into fragments of 300-800 base pairs, using a nebuliser
    2. these lengths are then degraded into a single-stranded DNA (ssDNA)
  • these are the template DNAs and are immobilised
    3. a sequencing primer is added and the DNA is then incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase and the substrates adenosine 5’ phosphosulfate (APS) and luciferin
  • only one of the four possible activated nucleotides, ATP, TTP, CTP, GTP is added at any one time and any light generated is detected

4.

  • one activated nucleotide, such as TTP is incorporated into the complementary strand of DNA
  • as this happens, two extra phosphoryls are released as pyrophosphate (PPi)
  • in the presence of
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

How many genes are there in the human genome?

A
  • 24,000
  • discovered after the Human Genome Project was finished in 2003
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

What does comparing human genomes and the genomes of other species tell us?

A
  • only a few human genes are unique to us
  • genes that work well tend to be conserved by evolution
  • as evolution progresses, some genes are c-opted to perform new tasks in each species
  • some changes to the regulatory regions of DNA that do not code for proteins have changed the expression of genomes
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

What do genome-wide comparisons tell us about evolutionary relationships?

A
  • has helped confirm evolutionary relationships between species
  • also has led to new knowledge about the relationships
  • DNA from bones and teeth of extinct animals can be amplified and sequenced
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

What do genome-wide comparisons tell us about variation between individuals?

A
  • about 0.1% of our DNA is not shared with other humans
  • this is around 3 million places on the DNA base sequence
  • the place where substitutions occur is called single nucleotide polymorphisms (SNPs)
  • some have no effect on the protein, some alter the protein, or the way RNA regulates expression
  • methylation also plays a major role - epigenetics!
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

How can sequenced genomes help predict amino acid sequences of proteins?

A
  • if the genome is sequenced, scientists can use the base triplet code to figure out which amino acids there are
  • this can determine the primary structure of proteins
  • researchers need to know which part of the gene codes for exons and introns
28
Q

What is synthetic biology?

A
  • an interdisciplinary science concerned with designing and building useful biological devices and systems
  • encompasses biotechnology, evolutionary bio, molecular bio, systems bio, biophysics
29
Q

What are some application of synthetic biology?

A
30
Q

How is DNA profiling done?

A
  • DNA is obtained from the individual: can be through blood, hair etc
  • DNA is broken down with restriction enzymes
  • these enzymes cut the DNA at specific recognition sites
  • the cut fragments will vary in size
  • fragments are separated by gel electrophoresis and stained
  • larger fragments travel the shortest distance
  • a banding pattern can be seen
  • the DNA which is being compared to is treated to the same method
  • banding patterns are then compared
31
Q

What was the former method of DNA analysis?

A
  • initially used restriction fragment length polymorphism analysis
32
Q

What is the current type of DNA used for analysis?

A
  • short tandem repeat sequences (STR)
  • they are highly variable short repeating lengths of DNA
  • the exact number of STRs varies from person to person
33
Q

How are STR sequences analysed?

A
  • STR sequences are separated by electrophoresis
  • each STR is polymorphic, but the number of alleles in the gene pool is small
  • thirteen STRs are analysed simultaneously
  • the chances of two people sharing STR sequences on all loci is 1 x 1018
  • the technology is very sensitive and even a trace of DNA left can produce a result
  • DNA can be stored for many years if a crime is unsolved
34
Q

What are some applications of DNA profiling?

A

Forensic Science:

  • establish innocence and convictions of criminals
  • identified Nazi war criminals in South America
  • identify remains of Richard III
  • identify victims’ bodies after crashes etc

Maternity and Paternity Disputes:

  • Half of the short tandem repeat Fragments come from the mother and the other from the father

Analysis of disease:

  • protein electrophoresis can detect the type of haemoglobin present and aid diagnosis of sickle cell anaemia
  • the number of sequences of Huntington disease can be detected by electrophoresis
35
Q

What is the definition of polymerase chain reaction?

A
  • a biomedical technology in molecular biology that can amplify a short length of DNA to thousands of millions of copies
36
Q

What basis does PCR rely on to work?

A
  • DNA is made of two antiparallel 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
37
Q

How is PCR different from DNA replication?

A
  • only short sequences of up to 10,000 base pairs can be replicated, not entire chromosomes
  • 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
38
Q

Describe the stages of PCR?

A
  • the sample of DNA is mixed with DNA nucleotides, primers, magnesium ions and the enzyme Taq DNA polymerase
  • mixture is heated to around 94-96°C to break H bonds between complementary nucleotide base pairs
  • this denatures the double-stranded DNA into two single strands of DNA
  • mixture is cooled to around 68°C to allow primers to anneal (by H bonding) to one end of each single strand of DNA
  • this gives a small section of double-stranded DNA at the end of each single-stranded molecule
  • the Taq polymerase enzyme molecules can now bind to the end where there is double-stranded DNA
  • Taq polymerase is obtained from a thermophilic bacterium, so its optimum is 72°C
  • the temperature is raised to 72°C, which keeps the DNA as single strands
  • the Taq DNA polymerase catalyses the addition of DNA nucleotides to the single-stranded DNA molecules, starting at the end with the primer and in the 5’ to 3’ direction
  • when the Taq DNA polymerase reaches the other end of the DNA molecule, a new double strand of DNA has been generated
  • the whole process begins again and is repeated for many cycles
  • amount of DNA increase exponentially
39
Q

What are the applications of PCR?

A
  • tissue typing: donor and recipient tissues can be types prior to transplantation to reduce risk of rejection
  • detection of oncogenes
  • detecting mutations
  • identifying viral infections: sensitive PCR tests can detect small quantities of viral genome - can help verify HIV or hep C
  • monitoring the speed of infectious disease
  • forensic science: small quantities of DNA can be amplified for DNA profiling
  • Research: amplifying ancient sources
40
Q

What is the definition of electrophoresis?

A
  • the process used to separate proteins or DNA fragments of different sizes
  • it can separate fragments that can differ by only one base pair
  • used in gene technology to separate DNA fragments for identification and analysis
41
Q

How does separating DNA by electrophoresis work?

A
  • it uses an agarose gel plate covered by a buffer solution
  • electrodes are placed in each end of the tank so that an electric current can pass through the gel
  • DNA has an overall negative charge due to the phosphate groups, so the fragments migrate towards the anode
  • fragments of DNA all have a similar surface charge
42
Q

How does separating proteins by electrophoresis work?

A
  • the principle is the same as separating DNA fragments but is often carries out in the presence of a charged detergent such as sodium dodecyl sulfate (SDS)
  • this equalises the surface charge on the molecules and allows proteins to separate as they move through the gel, according to Mr
43
Q

What is a DNA probe?

A
  • a short (50-80 nucleotides) single-stranded length of DNA that is complementary to a section of the DNA being investigated
44
Q

What may the DNA probe be labelled using?

A
  • a radioactive marker: usually with 32P in one of the phosphate groups in the probe strand
  • once the probe has annealed by complementary base pairing, to the piece of DNA, it can be revealed by exposure to photographic film
  • a fluorescent marker: emits the colour on exposure to UV light
  • fluorescent marker may also be used in automated DNA sequencing
45
Q

What are DNA probes useful for?

A
  • locating specific DNA sequences
  • to locate a specific gene needed for use in genetic engineering
  • to identify the same gene in a variety of different genomes from different species when conducting gene comparison studies
  • to identify the presence or absence of a specific allele for a particular genetic disease
46
Q

What is a DNA microarray used for?

A
  • placing a number of different probes on a fixed surface
  • it can reveal the presence of mutated alleles that match the fixed probes because the sample DNA will anneal to any complementary fixed probes
47
Q

Briefly explain how DNA microarrays work

A
  • sample DNA must first be broken into smaller fragments and may also be amplified using PCR
  • DNA microarray can be made using fixed probes, specific for certain sequences found in mutated alleles that cause genetic diseases, in the well
  • reference and test DNA samples are labelled with fluorescent markers
  • if a test subject and a reference marker both bind to a particular probe, the scan reveals fluorescence of both colours, indicating the presence of a particular sequence in the test DNA
48
Q

What are the potential benefits and hazards of genetically modifying microorganisms?

A
49
Q

What are the potential benefits and hazards of genetically modifying plants e.g. Bt tobacco and Bt maize?

A
50
Q

What are the potential benefits and hazards of genetically modifying soya beans

A
51
Q

What are the potential benefits and hazards of genetically modifying Golden Rice

A
52
Q

What are the potential benefits and hazards of genetically modifying plantains

A
53
Q

What are the potential benefits and hazards of genetically modifying crop plants resistant to pests

A
54
Q

What are the potential benefits and hazards of genetically modifying pathogens

A
55
Q

What are the potential benefits and hazards of genetically modifying mice

A
56
Q

What are the potential benefits and hazards of genetically modifying pharmaceutical proteins

A
57
Q

What are the potential benefits and hazards of genetically modifying silk

A
58
Q

What is germ line gene therapy?

A
  • gene therapy by inserting functional alleles into gametes or zygotes
  • their offspring may inherit the foreign alleles
  • has the potential to change the genetic makeup of many people, without consent
59
Q

What is somatic cell gene therapy?

A
  • gene therapy by inserting functional alleles into body cells
  • it affects only certain cell types
  • the alterations made to the patient’s genome in those cells are not passed to the patient’s offspring
60
Q

Describe how liposomes can be used in somatic cell therapy for cystic fibrosis

A
  • patients with cystic fibrosis lack a functioning CFTR gene
  • the alleles can be packaged within small spheres of lipid bilayer to make liposomes
  • these are placed into an aerosol inhaler and sprayed into the noses of patients
  • some will pass through the plasma membrane of the cells lining the respiratory tract
  • if they also pass through the nuclear envelope and insert into the host genome, the host cell will express the CFTR protein, which is a transmembrane chloride ion channel
  • epithelial cells in the respiratory tract are replaced every 10-14 days, so the treatment has to repeated at regular short intervals
61
Q

How can viruses be used for somatic cell therapy?

A
  • viruses have been used as vectors
  • a virus that usually infects humans is genetically modified so that it encases the functioning allele to be inserted
  • at the same time, it is unable to cause a disease but also enter recipient cells
  • in 1999 a patient died and in 2002 some patients developed leukaemia
62
Q

What are some potential problems using viruses as gene delivery agents for somatic cell therapy?

A
  • viruses, even if not virulent, may still provoke an immune or inflammatory response in the patient
  • patient may become immune to the virus making subsequent deliveries difficult or impossible
  • virus may insert the allele into the patient genome in a location that disrupts a gene involved in regulating cell division, increasing risk of cancer
  • virus may insert the allele into the patient’s genome in a location that disrupts the regulation of the expression of other genes
63
Q

How can artificial chromosomes be used in somatic cell therapy?

A
  • research is carried out into the possibility of inserting genes into an artificial chromosome that would co-exist with the other 46 chromosomes in the target cells
64
Q

What are some concerns over germ line gene therapy?

A
  • changes genetic makeup of offspring without giving consent
  • concern over how genes may be inserted
  • may find their way into a location that could disrupt the expression or regulation of other genes or increase risk of cancer
65
Q

Why is germ line gene therapy currently not allowed?

A
  • strict guidelines drawn up by regulatory bodies and ethics committees consider germ line therapy for humans to be ethically impermissible

A Level Biology (25 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions