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A2 Biology > 19.1 - PRINCIPLES OF GENETIC TECHNOLOGY > Flashcards

19.1 - PRINCIPLES OF GENETIC TECHNOLOGY Flashcards

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1
Q

State the 4 bases in a DNA

A

Adenine
Thymine
Guanine
Cytosine

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2
Q

What is a codon?

A

A sequence of 3 adjacent nucleotides in mRNA that codes for one amino acid

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3
Q

What does it mean when genetic code is universal?

A
  • every organism uses the same 4 bases
  • same codons will code for the same amino acids in all living things
  • genetic information is transferable between species
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4
Q

Define recombinant DNA

A
  • DNA that has been artificially changed by combining lengths of nucleotides from different species
  • DNA is altered and is introduced to new nucleotides –> recombinant DNA rDNA
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5
Q

What is a transgenic organism?

A

An organism that contains nucleotide sequences from a different species

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6
Q

What is a genetically modified organism? (GMO)

A
  • Any organism that has introduced genetic material

- Contain recombinant DNA

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7
Q

Why can recombinant DNA be easily formed?

A
  • Genetic code is universal
  • All living species uses the same bases
  • Same codons would code for the same amino acids
  • Genetic material is then transferable between species
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8
Q

Explain what is genetic engineering

A
  • A technique used to deliberately modify a specific characteristic of an organism
  • involves removing a gene with the desired characteristic from one organism
  • and then transferring the gene using a vector into another organism where the desired gene is then expressed
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9
Q

Describe what steps must be taken for an organism to be genetically engineered

A

> identification of the desired gene
isolation of the desired gene by:
- cutting from a chromosome using enzymes (restriction endonucleases)
- using reverse transcriptase to make a single strand of complementary DNA (cDNA) from mRNA
- creating gene artificially using nucleotides
multiplication of gene
- using polymerase chain reaction (PCR)
transfer into organism using vector (plasmids)
- identification of cells with new gene (using marker) then clone

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10
Q

What is needed for genetic engineering ?

A 
> enzymes
- restriction endonucleases
- reverse transcriptase
- DNA polymerase 
- DNA ligase
> vectors
- used to deliver genes into a cell
- such as plasmids, viruses, liposomes 
> markers 
- genes that code for identifiable substances that can be tracked 
- such as GFP which fluoruses under UV light
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11
Q

State the 3 ways to isolate the desired gene

A

1) extracting gene from the DNA of the donor organism using restriction endonucleases enzymes
2) Use reverse transcriptase to synthesise a single strand cDNA from mRNA of the donor organism
3) synthesising the gene artificially using nucleotides

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12
Q

Describe extraction of genes

A
  • extracting gene containing the desired nucleotide sequence from the donor organism using restriction endonucleases
  • there are many different restriction endonucleases as they bind to a specific restriction site (specific sequences of bases) on the DNA
  • restriction endonucleases will separate the two strands of DNA at the specific base sequence by cutting the sugar phosphate backbone unevenly to give sticky ends (blunt if cut straight across)
  • sticky ends allow easy insertion of desired gene into the other organism’s DNA as hydrogen bonds will easily form with the complementary base sequences on the other DNA that has been cut with the same restriction enzyme
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13
Q

Describe gene isolation using mRNA and reverse transcriptase

A
  • use the mRNA that was transcribed for that desired gene
  • mRNA is combined with a reverse transcriptase enzyme where nucleotides create a single strand of complementary DNA (cDNA)
  • mRNA is used as template for formation of cDNA with reverse transcriptase
  • DNA polymerase is used to convert single strand cDNA into double-stranded DNA molecule with the desired code for the gene
  • advantageous: easy to find gene as specialised cells make specific mRNA where it does not contain introns
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14
Q

Describe artificial synthesis for gene isolation

A
  • computers used to generate the desired nucleotide sequence
  • short fragments of DNA are produced then joined to form longer sequences of nucleotides
  • it is the inserted into vectors (plasmid)
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15
Q

Explain the roles for restriction endonucleases enzyme in the transfer of a gene into an organism

A
  • to isolate the desired gene and separating DNA of vector for desired gene to be spliced into
  • by separating the two strands of DNA at specific base sequence by ‘cutting’ the sugar-phosphate base backbone in an uneven way to create sticky ends
  • easy insertion to a vector as hydrogen bond forms with the complementary base sequences on the other DNA that have been cut with the same restriction enzyme
  • many different restriction endonucleases as it binds to specific restriction sites (specific sequences of the bases in DNA)
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16
Q

Explain the role of reverse transcriptase

A
  • to produce single strand cDNA that contains the code for the desired gene
  • mRNA + free nucleotides –> cDNA with reverse transcriptase
  • mRNA used as template to synthesise cDNA
  • easy as mRNA does not contain introns and desired gene is easy to find
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17
Q

Explain the role of DNA polymerase

A
  • to convert the single strand cDNA into a double-stranded DNA molecule
  • enzyme builds second strand by pairing free nucleotides with complementary bases on cDNA strand
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18
Q

Explain the role of DNA ligase

A
  • catalyses the formation of phosphodiester bonds in the DNA sugar-phosphate backbone
  • allows isolated desired gene to be spliced into the vector (plasmid) to be transferred to the new organism
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19
Q

What is a plasmid?

A
  • small, circular rings of double-stranded DNA
  • naturally occur in bacteria
  • able to self replicate
20
Q

Explain the role of plasmid in gene transfer

A
  • plasmid is used to transfer the desired gene to a new organism
  • desired gene is inserted into the plasmid
  • plasmid is cut open using the same restriction endonuclease that was used to isolate the gene
  • therefore, plasmid and desired gene fragment have complementary sticky ends (hydrogen bonds formed between complementary bases)
  • DNA ligase forms phosphodiester bonds between sugar-phosphate backbone of DNA fragment and the plasmid to form –> recombinant plasmid that contains the desired gene
  • marker genes is inserted to the plasmid so cells containing recombinant plasmid can be identified
  • plasmid transferred to host cells (bacteria) by transformation process
  • transformed bacteria will be identified by the markers
21
Q

Keywords when explaining about plasmid vector preparation

A
  • restriction endonucleases (cut plasmid/ isolate gene)

- DNA ligase (forms phosphodiester bonds between sugar-phosphate backbone of plasmid and the gene fragment

22
Q

Explain what is a promoter and how it activates regulates/activates gene expression

A
  • a region of DNA (non-coding) that determines which gene will be expressed and thus regulate gene expression
  • it is the site where RNA polymerase binds to begin transcription
  • promoter ensures RNA polymerase recognise DNA template strand
  • promoter contains the transcription start (first nucleotide to be transcribed) and this is where the RNA polymerase enzyme will bind
23
Q

Why must promoter be added in the plasmid

A
  • to ensure the desired gene is expressed, an appropriate promoter is added
  • promoter will activate the gene expression
  • RNA polymerase binds to the promoter that contains the transcription start
24
Q

Describe a genetically engineered plasmid

A
  • contains the desired gene which has been cut with the same restriction enzyme that cut the plasmid vector
  • contains markers to identify the desired gene/ organisms with the rDNA
  • contains promoter for gene regulation and for gene to expressed
25
Q

What is a marker

A
  • a gene that is transferred with the desired gene
  • to identify which cells have been successfully altered/transformed
  • and contain the recombinant DNA
26
Q

What is used as markers?

A
  • fluorescent
  • due to green fluorescent protein (GFP)
  • GFP gene and desired gene are linked to specific promoter
  • when promoter is activated and gene is expressed, the recombinant bacteria can be detected when they glow green under exposure of UV light
27
Q

Why fluorescent genes as markers are preferable?

A 
> easy to identify
- fluoresces under UV light 
> economical 
- antibiotics is not needed 
- therefore, bacteria does not need to be grown on plates of agar containing ab 
> no risk of ab resistance 
- ab resistance won’t be passed onto another bacteria
- ab will not be less effective
28
Q

Why ab resistant genes are not favourable as marker genes?

A

> ab resistant genes may be accidentally transferred to other bacteria
- when spread, antibiotics may be less effective
spread caused by:
- conjugation (transfer of genetic material)
- transduction (transfer of genetic material via a virus)

29
Q

Describe the method of using ab resistance genes as marker genes

A

method:
- plasmid contains: desired gene, specific ab resistant genes and promoter
- bacteria are grown on agar plates containing the antibiotic
- bacteria containing the recombinant plasmids would be identified as it grows on the agar plate

30
Q

What is gene editing?

A
  • genetic engineers alter the DNA of organisms by inserting, deleting or replacing the DNA at specific sites in the genome known to cause disease
  • it is a form of genetic engineering where foreign DNA is not introduced into the genome
31
Q

Describe the new gene editing technique

A
  • CRISPR (clustered regularly interspaced short palindromic repeats)
  • technique involved using the natural defense mechanism of bacteria
  • evolved bacteria cut the DNA strands at a specific point determined by a guide RNA attached to an enzyme (Cas9)
  • once cut, scientists either insert, delete, or replace the faulty DNA with normal DNA
    > predesigned gene –> inserted
    > replaced
    > deleted gene results in frameshift mutation
  • repair enzyme catalyses the process
32
Q

What is PCR (polymerase chain reaction) used for?

A
  • used to produce large quantities of specific fragments of DNA/ RNA from very small quantities
  • to have billions of identical copies of the DNA or RNA sample within a few hours
33
Q

Where is PCR carried out?

A
  • PCR instrument –> thermal cycler

- automatically provides the optimal temperature for each stage and controls the length of time spent at each stage

34
Q

What is required in a PCR reaction?

A

> target DNA or RNA to be amplified
primers (forward and reverse)
- short sequences of single-stranded DNA that have base sequences complementary to the 3’ end of the DNA/RNA being copied
- they define the region to be amplified by identifying to the DNA polymerase where to begin building the new strands
DNA polymerase
- enzyme used to build the new DNA or RNA strand
- Taq polymerase is used as it does not denature at high temp
- optimum temp is high enough to prevent annealing of the DNA strands that have not been copied yet
free nucleotides
- used in the construction of the DNA/RNA strands
buffer solution
- to provide the optimum pH for the reactions to occur in

35
Q

Describe the 3 stages of PCR

A

1) Denaturation
- the double-stranded DNA is heated to 95 deg Celsius
- this breaks down the H bonds that bonds the two DNA strands together

2) Annealing
- temperature is decreased to between 50 - 60 deg so primers(forward and reverse) can anneal to the ends of the single strands of DNA

3) Elongation/ extension
- temperature is increased to 72 deg for a minute
- this is the optimum temp for Taq polymerase to build the complementary strands of DNA to produce the 2 new identical double-stranded DNA molecules
* see digram on sme

36
Q

What is the role of Taq polymerase

A
  • the enzyme used to build new DNA/RNA strand from free nucleotides and the separated strands as template
  • it is used as it does not denature at high temperature in the first stage of PCR
  • optimum temperature is high enough to prevent annealing of the DNA strands that have not been copied yet
37
Q

What is gel electrophoresis?

A
  • it is a technique used widely in the analysis of DNA, RNA and proteins
  • during electrophoresis, the molecules are separated according to their size/mass and their net charge
38
Q

Why does separation occur in gel electrophoresis?

A

> due to the electrical charge the molecules carry
- +ve charged molecules move towards the cathode (-ve pole) and otherwise
- DNA is -vely charged due to the phosphate groups, therefore it will move towards the anode when placed in an electric field
different size
- different sized molecules will move through the gel at different rates
- gel has tiny pores –> small molecules move quickly and whereas larger molecules move slowly
type of gel used
- different gels will have different sized pores
- this would affect the speed the molecules can move through them

39
Q

Describe how to prepare DNA fragments so that it can be analysed for genetic profiling

A
  • DNA collected from anywhere on the body
  • To prepare the DNA fragments, the number of DNA molecules are amplified by PCR
  • restriction endonucleases are used to cut the DNA into fragments
  • different restriction endonucleases will cut the DNA at different base sequences
    scientists use enzymes that cut close to the variable number tandem repeats (VNTR) regions
  • VNTR are regions found in the non-coding part of DNA –> contain variable numbers of repeated DNA sequences that vary between different people (except identical twins)
40
Q

Describe how to separate the DNA fragments in gel electrophoresis

A
  • create an agarose gel plate in a tank
  • wells (series of groves) are then cut into the gel at one end
  • submerge gel in an electrolyte solution in the tank
  • insert fragments into the wells using a micropipette
  • apply electrical current to the tank
  • negative electrode connected to the end of the plate with the wells as DNA fragments will move towards the anode
  • smaller and shorter piece of DNA fragments will move faster and further from the wells than larger fragments
  • fragments are not visible –> therefore transferred onto absorbent paper which is heated to separate the two DNA strands
  • probes are added, an X-ray image is taken, or UV light is shone onto the paper, producing a pattern of bands
  • it is compared to a control fragment of DNA
41
Q

What are probes? (used in DNA separation)

A
  • used in DNA separation in gel electrophoresis to make fragments visible
  • probes are single-stranded DNA sequences that are complementary to the VNTR region
  • probes are used to be identified
    > a radioactive label (phosphorus isotope) whic causes probes to emit radiation that makes X-ray film go dark, creating a pattern of dark bands
    > fluorescent stain (ethidium bromide) which fluoresces when exposed to UV, creating a pattern of coloured bands
42
Q

What are microarrays?

A
  • laboratory tools used to detect the expression of thousands of genes at the same time
  • and to identify the genes present in an organism’s genome
  • large number of genes can be studied in a short period of time
  • small piece of glass/ plastic/ silicon that have probes attached to a spot (gene spot)
  • probes –> complementary to a specific base sequence, depending on the purpose of the microarray
43
Q

Explain how microarray is used to analyse genomes

A
  • DNA is collected from the species
  • restriction enzymes are used to cut the DNA into fragments
  • these fragments are denatured to create single-stranded DNA molecules
  • fragments are labelled using fluorescent tags (fragments from different sources are tagged in different colours)
  • fragments hybridise with the probes on the microarray
  • after a set period of time, DNA that was not hybridise with the probes is washed off
  • microarray is examined using UV light which causes the tags to fluoresce
  • or microarray is scanned (colours are detected) by a computer and information is analysed and stored
  • presence of colour indicates where hybridisation has occurred –> DNA fragment is complementary to the probe
  • if red or green fluoresces, only one species of DNA had hybridised
  • if yellow, both species have hybridised with the DNA fragment
  • if spot lacks colour, the gene is not present in either species
44
Q

Describe how microarrays are used to compare which genes are being expressed

A
  • when gene are being expressed, many copies of mRNA is produced by translation
  • corresponding proteins are produced from mRNA during translation
  • microarrays are used to detect whether a gene is being expressed

method:
- mRNA is collected from both types of cells
- reverse transcriptase is used to convert mRNA into cDNA
- PCR may be used to increase the quantity of cDNA
- fluorescent tags are added to cDNA
- cDNA is denatured to produce single-stranded DNA
- single-stranded DNA is allowed to hybridise with the probes on the microarray
- UV is shone on the microarray, spots that fluoresce indicate gene was transcribed
- intensity of light indicates quantity of mRNA produced

45
Q

What are the types of data being collected from analysing genes using technologies such as microarrays and gene sequencing

A
  • sequences of genome when genes are being expressed

- structure (sequence of amino acids) and functions of proteins

46
Q

State the benefits of using databases that provide information about nucleotide sequences of genes and amino acid sequences of protein

A

> allows scientists to make comparisons with genomes of other organisms using the databases available
- help to find the degree of similarity between organisms and indication how closely related the organisms are
- indicate whether there are organisms that could be used in experiments as a model for humans
use database with the genome of Plasmodium
- to determine which genes/ proteins could be altered or affected to control the parasite
- e.g: finding vaccine for malaria

A2 Biology (3 decks)

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