Gene Technologies Flashcards
(51 cards)
1
Q
What is Recombinant DNA
A
- form of genetic engineering
- involves the transfer of fragments of
DNA from one organism to another via isolating fragments of interest
2
Q
Why is genetic information transferable between species?
A
Genetic code is universal therefore the same amino acid code for the same amino acids in all living things
3
Q
What are the 3 methods to create fragments of DNA?
A
- Reverse Transcriptase (enzyme)
- Restriction endonucleases (enzyme)
- Gene machine
4
Q
What is Reverse Transcriptase?
A
This process uses reverse transcriptase to make DNA copies of mRNA that was transcribed from the gene of interest
5
Q
What is the process of Reverse Transcriptase?
A
- A cell that naturally produces protein of interest is selected
- Once mRNA is isolated reverse transcriptase joins DNA nucleotide w/ complementary base pairs to the mRNA sequence
- the mRNA is used as template to make the single stranded complementary DNA
- DNA polymerase is then used to convert the single stand of cDNA into double stranded which contains the desired code for the gene
6
Q
Advantage of Reverse Transcriptase
A
- cells containing protein of interest make large amounts of mRNA → so lots of the mRNA of interest is available to make cDNA
- introns are already removed→ so cDNA doesn’t include introns, prokaryotes cant remove introns so not an issue
7
Q
Disadvantage of Reverse Transcriptase
A
- more steps, so more time consuming & more difficult
8
Q
What is restriction endonucleases?
A
- this process uses the enzyme restriction endonucleases to cut up the DNA
- RE naturally occur in bacteria as a defence mechanism (if there is foreign DNA inside a bacteria, the enzyme will cut it up so it doesn’t replicate and cause harm)
9
Q
What is the process of restriction endonucleases?
A
- RE will separate the 2 strands of DNA at a specific base sequence by cutting the sugar-phosphate backbone to give sticky ends or **blunt ends **
BLUNT ENDS:
- caused be being cut straight across
- do NOT result in exposed bases
STICKY ENDS
- caused by cutting the DNA staggered
- result in exposed DNA bases
- palindromic (read the same front to back) - At sticky ends, the exposed base pairs can be aligned to exposed bases of the organism you want to insert it in → makes it easier to insert the desired gene into another organisms DNA
can easily form H bonds w/ complementary base sequences on other pieces of DNA that have been cut w/ same restriction enzyme
10
Q
Advantage of Restriction Endonuclease
A
- Sticky ends on DNA fragment make it easier to insert in the DNA fragment when creating recombinant DNA
- can select enzymes that’ll cut at palindromic recognition sites to give sticky ends
11
Q
Disadvantage of Restriction Endonucleases
A
- Still contains introns as its cut from the original DNA–> an extra step is required to remove introns
12
Q
What is the gene machine?
A
using computers to generate the nucleotide sequence to produce the gene
13
Q
What is the process of the gene machine?
A
- Scientists examine the protein of interest to identify the amino acid sequence
- The DNA sequence is entered into the computer (this checks that he DNA being created is safe and ethical to produce)
- The computer can create small sections of overlapping single strands of nucleotides that make up the gene (aka oligonucleotides)
- Oligonucleotides can then be joined to create the DNA for the entire gene
- Polymerase Chain Reaction can be used to make lots of copies of the gene and make it double stranded
14
Q
Advantages of Gene Machine
A
- very quick, very accurate
- can design exact DNA fragment you want with sticky ends, DNA probes & won’t have introns
15
Q
Disadvantages of Gene Machine
A
- need to know the sequence of amino acids or bases
- time consuming
16
Q
what is in vivo cloning?
A
a way to amplify the DNA fragments which have been isolated by recombinant DNA technologies inside of a living thing
17
Q
the stages of insertion of DNA fragment into a vector
A
- promoter region and terminator region
- cutting
- gluing
18
Q
what is a promoter region?
A
- short sequence of DNA bases & is the binding site for RNA polymerase to enable transcription to occur
- added at the start of the DNA fragment so RNA polymerase can attach and transcription can occur
19
Q
what is a terminator region?
A
- causes RNA polymerase to detach at the end of gene and stop transcription, ensuring one gene at a time is copied into mRNA
- added at the end of the gene so RNA polymerase can detach and transcription stops
20
Q
what happens if the promotor region doesn’t attach at start of gene?
A
- RNA polymerase cant attach
- transcription cant occur
21
Q
what is a vector?
A
something that can carry the isolated DNA fragments into the host cell
- commonly formed from bacteria plasmids
22
Q
Describe the process of inserting DNA into a vector
A
CUTTING
1. restriction endonuclease enzyme cuts open the plasmid
( same RE used to cut open the DNA plasmid)
2. DNA fragments sticky ends (exposed nucleotides) are complementary to sticky ends of plasmid so attach easily forming recombinant plasmid
GLUING
3. ligase anneals the cut plasmid and DNA fragment
4. Ligase catalyses the condensation reaction to form phosphodiester bonds between nucleotides
23
Q
Describe the process of getting the vector into the host cell
A
- the vector (plasmid w/ recombinant DNA) can only enter the host cell if the cell membrane is permeable
- Calcium ions and a sudden increase in temp, increases fluidity of cell membrane of host cell
- this enables vector to end host cells cytoplasm
24
Q
3 issues that can occur that mean the host cell hasn’t successfully taken up the recombinant plasmid
A
- recombinant DNA doesn’t get inside the cell even after increasing permeability of cell membrane
- the plasmid re-joins with itself before the DNA fragment can combine
- the isolated DNA fragment sticks to itself, rather than inserting into the plasmid
25
what are marker genes?
used to identify which bacteria successfully took up the recombinant plasmid
26
3 types of marker genes
1. antibiotic resistance genes
2. genes coding for fluorescent proteins
3. genes coding for enzymes
27
describe how antibiotic resistant marker genes are used
- gene for resistance to tetracycline is used.
- bacteria which have their gene cut that produces enzyme that breaks tetracycline down, are identified.
- this is done by growing them on a culture containing tetracycline.
28
describe how fluorescent markers are used
1. green fluorescent protein gene is inserted into the bacteria plasmid
2. isolated DNA fragment deliberately inserted in the middle of marker gene
3. bacteria is grown on petri dish
4. expose the colonies to UV light and those with GFP gene will glow ( non- glowing colonies contain recombinant plasmid as GFP gene has been disrupted)
29
describe how enzyme marker are used (lactase)
lactose can turn a substance from blue to colourless
1. the lactase gene is inserted into the plasmid
2. isolated DNA fragment inserted in the middle of this gene
3. this prevents lactase production SO if substance remains colourless, recombinant plasmid must've attached
30
why does the DNA fragment need to be inserted in the middle of the gene?
to disrupt the gene
31
how do we make multiple copies of the host cell that has recombinant plasmid of interest?
fermenter is used to make copies to produce large abundance of genes of interest
32
what is in vitro cloning?
cloning of the DNA fragments that have isolated by recombinant DNA inside a not living thing
- This is done by the polymerase chain reaction
33
describe the process of the polymerase chain reaction
1. the temperature is increased to **95 degrees** to break the hydrogen bonds and split the DNA into 2 single strands
2. the temperature is then decreased to **55 degrees** to allow primers to attach (cool enough for H bonds to reform so primer can be held in place)
3. taq DNA polymerase then attaches to complementary free nucleotides. (temp remains at 72 degrees as this is optimal)
34
advantages of polymerase chain reaction
1. automated machine: more efficient as its not a complex process
2. rapid: huge amounts of copies of DNA fragments can be made at a fast rate
3. doesn't require living cells: quicker and less complex techniques in comparison to in vivo cloning
35
Polymerase cain reaction equipment list
- DNA fragments to be amplified
- Primers
- taq DNA polymerase ( found in bacteria has a much higher optimum temp than humans therefore wont denature in PCR)
- DNA nucleotides
- thermocycler
36
What is a DNA probe?
Short, single stranded prices of DNA with complementary base pairs that are labelled radioactively/ fluorescently to be identified
37
What makes DNA probes identifiable?
- Radioactively labelled probes
-Fluorescently labelled probes
38
what is DNA hybridisation?
mixing and binding of DNA probes and patients DNA to see if they're complementary
39
describe the process of DNA hybridisation
1. patients DNA sample is heated up to break H bonds between bases to make the DNA single- stranded
2. patients single stranded DNA is mixed w/ the DNA probe and cooled
3. complementary base sequences can align and form H bonds
40
use of gene techinolgies
- allows screening for potential genetic disorders
- can identify presence of oncogenes
- can screen for multiple disease simultaneously using an array where multiple DNA probes are present
41
How to locate alleles of genes?
1. must know DNA base sequence in order to design the probe
2. fragment of DNA can be produced using gene machine
3. DNA fragment can be amplified using in vitro or in vivo cloning
4. add label to DNA probe- radioactive nucleotide or fluorescent label
5. After hybridisation , DNA is washed so any unbound probes are washed away
42
why do we use gene technologies?
1. drug screening -some drugs are more/ less effective depending on genotype
2. cam help determine effective dose
- saves money
- increased effectiveness
- increase safety
-less waste
43
what is genetic screening?
method used to screen for potential genetic disorders or for the presence of oncogenes
44
advantage of genetic screening
- allows for medicine to be tailored to patients particular genotype
- allows individuals to be checked more regularly if a certain allele is present (e.g breast cancer allele)
45
cons of genetic screening
- if patients find out they have an inventible disease through alleles they posses this might impact how they life their life
- arises the issues of designer babies which is unethical
46
what are variable number tandem repeats- VNTRs
long sequences of DNA bases that repeat but don't code for anything
- genetic fingerprinting is the examination of VNTRs
47
stages of genetic fingerprinting
1. collection and extraction
2. digestion
3. separation
4.hydbridisation
5. development
6. analysis
48
describe the stages of genetic fingerprinting
1. small sample of DNA is collected and amplified by PCR
2. Restriction endonucleases cut the DNA into smaller DNA fragments just before and after the VNTR
3. gel electrophoresis separates the cut up VNTRs
4. DNA hybridisation: DNA probes added to the DNA in gel, gel is then rinsed to remove unbound DNA probes
5. VNTRs are transferred onto nylon sheet where is it exposed to UV light/ X-rays to see if probes attached
6. The position of the DNA bands are used to compare genetic relations
49
describe the process of gel electrophoresis
1. DNA sampled are loaded onto agar gel, a buffer liquid is applied and electrical voltage, end nearest to the DNA is negatively charged and end furthest from DNA is positively charged
2. agar gel creates resistance for the moving DNA, smaller VNTRs cam move faster and further along the gel than longer VNTRs
3. alkaline is then added to the gel to make the DNA single stranded in preparation for DNA hybridisation
50
what is the role of the marker in analysing gel electrophoresis results?
- marker is a DNA sample with VNTRs of known lengths
- can figure out what VNTRs a sample has by comparing to marker
51
Structures of proteins that allow them to be separated in gel electrophoresis
1. R groups
2. Charge
3. Mass
