6.3 Manipulating Genomes Flashcards
(185 cards)
1
Q
Define DNA sequencing.
A
A technique that allows genes to be isloated and read.
2
Q
What was FRed Sanger’s approach to Dna sequencing?
A
He used a single strand of DNA as a template for 4 experiments in separate dishes. Each dish contained a solution with 4 bases- A, T, C and G- plus an enzyme, DNA polymerase.
3
Q
What did Fred Sanger develop?
A
He developed a methord that ultimetly allowed scientists to sequence whole genomes.
4
Q
What did Sanger add to each dish when sequencing DNA?
A
To each dish, a modified version of one of the DNA bases was added. The base was modified in a way that, once inporperated into the synthesised compelmentary strand, no more bases could be added. Each modified base was also labelled with a radioactive isotope.
5
Q
In Sangers experiment to sequence DNA, what was the added modified DNA bases labelled with?
A
A radioactive isotope.
6
Q
In Sangers experimentz to sequence DNA, what did the modified DNA base cause once added to one of the dishes?
A
Once incorperated into the synthesised complementary strand of DNA, no more bases can be added.
7
Q
What is generated as Sangers experiment to sequence DNA progresses?
A
Thousands of DNA fragments of varying lengths were generated.
8
Q
In Sangers experimentz to sequence DNA, what happens to the DNA fragments? Why is this done?
A
The DNA fragments were passed through a gel by electropheresis. Smaller fragments travel further, so the fragments beome sorted by length.
9
Q
In Sangers experiment to sequence DNA, what would be the first base sequence of a DNA fragment be if the first one-base fragment had thymine at the end?
A
T
10
Q
What is this a picture of?
Previous was T
A
Fred Sangers experiment to sequence DNA.
11
Q
What is Fred Sangers methord for sequencing DNA like?
A
The methord is efficeint and safe.
12
Q
What did Sanger do with his methord to sequence DNA?
A
He used to to sequence the genome of a phage virus ( a virus that infecfts bacteria) called Phi-X174, the first DNA based organism to have its genome sequenced.
13
Q
How did Sanger find the gemone of Phi-X174?
A
He had to count the bases, one by one, from the bands in a piece of gel- a very time consuming and costly process.
14
Q
When cloning DNA, how isn a gene isolated?
A
By using restriction enzymes, from a bacterium.
15
Q
How is DNA cloned for DNA sequencing?
A
- The gene to be sequenced must be isolated, using restriction enzymes, brom a bacterium.
- The DNA was then instered into a bacterial plasmid (the vector) and then into a Eshchichia coil bacterium hosts that, when cultured, divides many times, enabling the plasmid with the DNA insert to be coppied.
- Each new bacterium contained a copy of the candidates gene. These lengths of DNA were isolated using plasmid preparation techniques and were then sequenced.
16
Q
When cloning DNA, how are multiples of the same single standed gene made?
A
The DNA is inserted into a bacterial plasmid (the vestor) and then into a Escherichia coil bactrium host that, when cultured, it divides many times, enabling the plasmid inserted to be coppied many times.
17
Q
When cloning DNA, how is the coppied DNA in each bacterium removed?
A
These lengths were isolated using plasmid preparation techniques and were then sequenced.
18
Q
Based on Fred Sangers method, what was developed in 1986?
A
The first automated DNA seqencing machine.
19
Q
What was different about the new automated DNA sequencing machine and Fred Sangers methord?
A
Flouresent dyes instead of radioactivity were based to label the terminal bases.
20
Q
What is an autroadiogram?
A
In the automated DNA sequencing machine, The flouesent dyes glowed when scanned with a laser beam, and the light signiture was identified by the computer. This methord dispensed with the need for technicians to read autoradiograms.
21
Q
What is pyrosequencing?
A
A methord developed in 1996 and uses sequencing by sythesis to carry out DNA sequencing.
22
Q
By what prosess was Sangers method of DNA sequencing?
A
Chain determination.
23
Q
What does the process of pyrosequencing involve?
A
It involves synthesising a single strand of DNA, complementary to the strand to be sequenced, one base at a time, whilst detecting, by light emission, which base was added at each step.
24
Q
What is the 1st step of pyrosequencing?
A
- A long strand of DNA to be sequenced is mechanically cut into fragments of 300-800 base pairs with a nebuliser.
25
What is the second step of pyrosequencing?
The lengths are then **degraded into single-stranded** DNA. These are the template DNAs and they are **immobalised**.
26
What is the 3rd stage of pryosequencing?
A **sequencing primer** is added and then the DNA is **incubated** with **enzymes** and different **substrate**s. Once **one of the 4** possible **activated nucleotides,** ATP, TTA, CTP and GTP is added at any one time and **any light** that is generated is detected.
27
During pryosequencing, WHat enzymes and substrates is the ssDNA incubates with?
* DNA polymerase
* ATP sulfurylase
* Luciferase
* Apyrase
* Adenosin 5' phosphosulfate (APS)
* Luciferin
28
What is happening is the 4th step, part a of pyrosequencing?
One **activated nucleotide** (nucleotide with 2 extra phosphoryl groups) such as TTP is, **incorperated into a complementary strand** of DNA using the strand to the sequenced as a **template**.
29
What is happening in the 4th step, part b of pyrosequencing?
As the activated nucleotide is incorperated into the complementray strand of DNA, the **2 extra phosphoryls are released as pyrophosphate** (PPi).
30
What is happening in the 4th step, part c of pyrosequencing?
In the presence of **APS,** the enzyme **ATP sulphurylase** converts the pyrophosphate into ATP.
31
What is happening in step 4, part d in pyrosequencing?
In the presence of ATP, the enzyme luciferase converts to oxyluciniferase.
32
What is happening in step 4, part e of pyrosequencing?
Converting luciferase to oxylucinefase **generates visable light** which can be detected by a camera.
33
At the end of pyrosequencing, what is the light generated proportional to? What does this tell us?
The amount of light generated is **proportional to the amount of ATP available at the end**, indiciating how many of the **same type of activated nucleotide**s were **incorperated adjecently** into the complementary DNA strand.
34
At the end of pyrosequencing, what happens to the unincorperated acivated nucleotides?
They are degraded by **apyrase** and the reaction starts again with a different nucleotide.
35
How fast is DNA sequenced in pyrosequencing?
1 million reads occur simultaneously, so a 20-hour run generates 400 million bases of sequencing information. Software packages assemble these sequences into longer sequences.
36
What is bioinformatics?
Science of collecting and **analysing complex biological data such as genetic codes**, e.g. DNA sequencing.
37
What has been specially designed to store and analyse bioinformatics?
Software packages.
38
What is the Human Genome Project?
An international project to chart the entire genetic material of a human being, completed in 2003.
39
How many genes do humans have, found in the Human Genome Project?
24,000
40
What does whole genome sequencing determine?
It determines the complete DNA sequence of an organisms genome.
41
In case of eukaryotic cells, what are stored in gene banks?
Sequenced genomes are stored in gene banks.
42
What is a edukarotes complete DNA sequence made up of?
The genetic material of the chromosomes, mitochondia and chloroplasts.
43
What became clear when the human genome was compared with those of other species?
A few human genes are unique to us.
44
We share 99% of our genes with chimpanzees, what does this tell us?
It shows that the **genes** that work well tend to be **conserved by evolution**.
45
What is an example which genes being conserved by evolution can be seen? How have we taken advangtage of this?
| Insulin
Pigs and humans have similar genes for insulin, which is why, prior to genetically-modifying bacteria to make insulin, pig insulin was used to treat patients with diabetes.
46
What does it mean if a gene is co-opted?
Co-option occurs when** natural selection finds new uses for existing traits**, including genes, organs, and other body structures.
47
What has happened to the gene FOXP2 in humans which makes us different from other mammals?
Tiny changes in the gene (co-option), means that in humans this gene allows speech.
48
Many differences between organisms are not because the organisms have totally differennt genes, what could this be because of?
Its because some of their **shared genes** have been **altered** and now work in a subtly different way.
Some changes to** regulatory regions** od DNA that do not code directy for proteins have also altered the **expression of genomes.**
49
How can changes in the regulatory regions of DNA alter the expression of genomes?
Regulatory and coding genes interact in such ways that, without increasing the number of genes, the **number of proteins made may be increased**.
50
Comparing genomes of different organims is good to look at relationships, what can this lead to?
Organisms being reclassified.
51
How can an extinct animals evolutionary relationship be verified?
The **DNA** from the bones and teeth of some extinct animals can be **amplified and sequenced**.
52
How may humans be genetically different?
Humans all have the **same genes**, but we have **different alleles**. 0.1% of our DNA is not shared with others.
53
All humans are genetically similar, what is an example of a rare exeption for this?
In rare cases, where a gene is lost by deletion of part of a chromsome.
54
Why is it significant that we dont share about 0.1% of our DNA with others?
Although 0.1% sounds small, our genome **contains 3 billion DNA base** pairs, this means that **3 million places** on the DNA length where our DNA sequences can **differ**, due to **mutations**, such as substitution.
55
What are the places on out DNA where substitutions occur called?
| Cauing variation
Single nucleotide polymorphisms (SMPs).
56
what is single nucleotide polymorphosisms (SNPs)?
Places on DNA where substitutions occur.
57
What is the effect of SNPs?
Some have **no effect** on the protein, some can alter a protein or alter the way a piece of** RNA regulates** the **expression** of another gene.
58
What process plays a major role in regulating gene expression in eukaryotic cells?
Methylation
59
What can methods to map mythelation of whole human genomes help researchers do?
Can help them understand the development of certain diseases, e.g. certain types of cancer and why they may or may not develop in genetically similar individuals.
60
What is epigenetics?
the study of **changes in organisms** caused by **modification of gene expression** rather than alteration of the genetic code itself, e.g. study of mythelation
61
Describe determining the sequence of animno acfids within a protein without having the organims genome.
Its a laborius and time consuming process.
62
How can researchers find the sequence of amino acids in a protein if they know the organims genome?
If they know the **organims genome** and know which codes for a **specific protein**, by using knowldge of which **base triplet code** for which amno acids, they can determine the **primary structure** of proteins. The researcherd need to know which part of the gene codes for **introns and exons.**
63
What is synthetic biology?
An **interdisciplinary science** concerened with **designing and building useful biological devises and systems**.
64
What sciences does synthetic biology concern?
* Biotenology
* Evolutionary biology
* Molecular biology
* Systems biology
* Biophysics
65
What may bge the ultmiate goals for synthetic biology?
To build engineered biological systems that store and process information, provide food, maintain human health and enhance the envitoment.
66
How is synthetic biology helped by DNA sequencing?
The sequences of DNA found by **analysing genomes** provides **potential building blocks** for synthetic biologists to **build devices**.
67
What are some examples of the application of synthetic biology?
* Information storage
* Production of medicines
* Novel proteins
* Biosensors
* Nanotechnology
68
What is the aplication of information storage from synthtic biology?
Scientists can **encode vast amounts of digital information** onto a **single strand of sythetic DNA**.
69
What is the aplication of production of medicines in synthtic biology?
*Escherichia coli* and yeast have both been **genetically engineered** to produce the **precoursor of a good anitmalarial drug**, arteminsinin, previously only available by exracting if from ceratin parts of the *Artemisia* plants at particular times in the plants life cycle.
70
What is the aplication of novel proteins in synthtic biology?
**Designed proteins** have been produced, e.g. **one that is similar** to haemoglobin and binds to oxygen, byt not carbon monoxide.
71
What is the aplication of biosensors in synthtic biology?
**Modified bioluminecsent bacteria**, placed on a coating microchip, glow if air is polluted with petroleum pollutants
72
What is the aplication of nanotechnology in synthtic biology?
**Material can be produced for nanotechnology**- e.g. amyloid fibres for making biofilms- for functions such as adhesion.
73
What is bioethics with synthetic biology?
Synthetic biology **raises issues of ethics and biosecurity**.
Synthetic biology is **not about making synthetic forms from scratch**, but is a **potential for new systems** with rewards and associated risks to be managed.
74
Define electrophoresis?
Process used to separate protein or DNA fragments to different sizes.
75
What is electrophoresis used for?
Use to separate proteins or DNA fragments of different sizes.
76
What does electrophoresis use?
It uses an agarose gel plate covered by a buffer solution.
77
What is a tandem repeat sequence?
Tandem repeats are repetitive segments of DNA that do not code for proteins.
78
How long can a tandem repeat sequence be?
Between 10 and 100 base pairs long.
79
What is a key feature of tandem repeat sequences?
They all feature the same core sequence.
80
Where do tandem repeats occur?
They occur at more than 1000 locations in the genome, and in each place, they may be repeated a random number of times.
81
Some types of tandem repeats are highly variable, what are they called?
Variable number tandem repeats (VNTRs).
82
What does the number of tandem repeats show?
The number of tandem repeats showed a family resemblance, but the DNA profile for each family member was unique.
83
What did Jefferys realise while looking at DNA profiling about a persons DNA profile?
A persons DNA profile could confirm or refute paternity and maternity.
84
What is the procedure of DNA profiling?
DNA is obtained from the individual.
DNA goes through PCR.
DNA is digested by restriction enzymes into short tandem repeats.
Fragments are separated by gel electrophoresis and stained.
Banding pattern can be seen.
DNA to which the individuals is being compared is treated with the same process. The banding pattern of the DNA samples can then be compared.
85
How is DNA collected for DNA profiling?
Either by mouth swab from saliva on a tooth brush, from blood or hair, bone from ancient remains.
86
In DNA profiling, how is the DNA strand split up?
The DNA is digested by restriction enzymes. These enzymes cut the DNA at specific recognition sites, either side of the short tandem repeats. The fragments will vary in size.
87
What is used for modern DNA analysis?
Short tandem repeats.
88
What did the first methord of DNA analysis involve?
It involved restriction length **polymorphism analysis**. Thia methord is laborious and is no longer used.
89
What are short tandem repeats?
These are highly variable short repeating lengths of DNA.
90
How does STRs vary from person to person?
STRs vary in length from person to person.
91
How are STR sequences separated?
By elecrophoresis.
92
What is the gene pool like for STRs?
Each STR is polymorphic , but the number of alleles in the gene pool for each one is small.
93
During DNA profiling, how many STR are analysed at one time?
13
94
What are the chances of 2 individuals sharing STR sequences at all the same loci?
1 x 10^8
95
Why is it unlikely to get 2 people sharing the dame STR sequences?
Chances 2 people have the same is 1 x 10^8- this is greater than the number of people on earth. However, there are an estimated 12 million identical twins on earth which would share them.
96
What can DNA profiling be used for?
Forensic science
Maternity and paternity disputes
Analysis of disease
97
Why must DNA samples to go through DNA profiling be treated carefully?
To ensure the sample is not comtaminated.
98
Regarding to DNA profiling and DNA samples, what can be done with a sample if a crime case is unsolved?
The DNA can be stored, then later be used to assess new evidence.
99
For what purposes has DNA profiling been used for in forensic science?
To identify was criminals hiding in South America.
To identify victims body parts after disasters.
To match profiles from descendants of those lost during WWI with the unidentified remains of the soldiers who fell in the battlefields in Northern France.
100
How can DNA profiling by used in maternity and paternity disputes?
Half of every childs genetic information comes from the mother and half comes from the father, so half the short tandem repeat fragments come from the mother and half come from the father. Comparing the DNA profiles of mother, father and child can therefore establish maternity and paternity.
101
How is DNA profiling used in the analysis of disease?
Protein elecrtophoresis can detect the type of haemoglobin present and aid diagnosis of sickle cell anaemia.
A varying number of STR sequences for a condition such as Huntington disease can be detected by elecrophoresis.
102
Define the polymerase chain reaction (PCR)?
A biomedical technology in molecular biology that can amplify a short length of DNA to thousands of millions of coppies.
103
What does PCR do?
PCR amplifies DNA so that it can be analysed.
104
What is PCR?
It is the artificial replication of DNA.
105
What does PCR rely on?
It relies on the facts:
DNA is made up of 2 antiparallel backbone strands.
Each strand of DNA had a 5' and and a 3' end.
Base pairs [air up according to complementary base pairing.
106
How does PCR differ from DNA replication?
Only short sequences, of up to 10,000 base pairs 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 DNA strands to the replicated.
107
What sort of reaction is PCR?
A cyclic reaction.
108
Primarily, why was PRC a time consuming process?
The DNA had to be heated to denature it and then cooled to about 35°C to anneal the primers and allow the DNA polymerase to work.
109
PCR was originally a time consuming process, how was this solved?
DNA polymerse was obtained from the thermophilic bacterium Thermophilus aquaticus. This enzyme called Taq polymerase and is stable at high temperatures.
110
What is the DNA polymerase enzyme called in PRC and where did it come from?
The enzyme is called Tac polymerase and it is from a thermophilic bacterium.
111
Why is Tac polymerase good for PCR?
The enzyme is stable at high temperatures.
112
What are the steps of PCR?
Sample of DNA is mixed with DNA nucleotides, prime, magnesium ions and Tac polymerase.
Mixture is heated ti turn the double strand to 2 single strands.
The mixture is cooled so primers can anneal to each single stand of DNA.
Tac DNA polymerase can now bind to the end where the primer is.
Temperature is raised to 72°C keeping the DNA single stranded.
Tac DNA polymerase catalyses the addition of DNA nucleotides, starting at the primer and proceeding in the 5' to 3' direction.
When Tac DNA polymerase reaches the other end, a new double strand of DNA has been generated.
Whole process begins again for many cycles.
113
Why in the 2nd step of PCR is the mixture of DNA heated? (what temperature)
Heated to about 95°C the hydrogen bonds between complementary base pairs are broken, therefore, denaturing the double-stranded DNA into 2 Single strands of DNA.
114
In the 1st step of PRC, what is the sample of DNA mixed with?
DNA nucleotides
Primers
Magnesium ions
Enzyme Tac DNA polymerase
115
In the 3rd step of PCR, what temperature is the mixture cooled to?
68°C
116
What is the optimum temperature for Tac polymerase?
72°C
117
What can PCR be used for?
Tissue typing
Detection of oncogenes
Detecting mutations
Identifying viral infections
Monitoring the spread of infectious disease
Forensic science
Research
118
How can PCR be used in tissue typing?
Donor and recipient tissues can be typed prior to transplant to reduce the risk of rejection of the transplant.
119
How can PCR be used in the detection of oncogenes?
If the type of mutation involved in a specific patients caner is found, then the medication may be better tailored to that patient.
120
How can PCR be used to detect mutations?
A sample of DNA is analysed for the presence of a mutation that leads to a genetic disease. Parents can be tested to see if they carry a recessive allele for a particular gene; fetal calls may be obtained by the mothers blood stream for parental genetic screening for parental genetic screening; during IVF treatment, one cell from an 8 cell embryo can be used to analyse the fetal DNA before implantation.
121
Define electrophoesis?
Elecrophoresis is the process used to separate proteins of DNA fragments of different sizes.
122
What equipment does electrophoresis require?
A gel plate covered by a buffer solution
Electrodes
123
How does the electric charge across the agarose gel cause the DNA fragments to move?
DNA has an overall negative charge, due to its many phosphate groups, so the DNA fragments migrate towards the anode (positive electrode).
124
Does the charge of DNA change depending on its size?
The charge on DNA doesnt really change depending on it size.
125
What must fist be done to the DNA samples before it can undergo elecophoresis?
DNA samples are digested with restriction enzymes to cut the, at specific recognition sizes, into fragments. This is carried out at 35-40°C and may take up to an hour.
126
How can you set up the elecrophroesis tank?
The agarose gel is made and poured into the central region of the tank, whilst the combs are in place at one end. Once the gel is set, buffer solution is added so that the gel is covered and the end sections of the tank contain buffer solution. Now the combs are carefully removed, leaving wells at one end of the gel.
127
In electrophoesis, before the DNA samples are added to the elecrophoresis tank, what is added to the digested DNA?
A loading dye is added to the tubes containing the digested DNA.
128
What is put into the wells in electrophoresis?
the digested DNA plus loading dye.
129
How is the digested DNA plus the loading dye put into the wells in the electrophoresis gel?
A pipette is used and this is held, in the buffer solution, just above one of the wells. The loading dye is dense and carries the DNA down into the well. The pipette should not be placed right into the well otherwise you might pierce the bottom of the well.
130
In electrophorsis, what do you do after you have loaded all the wells with different DNA samples?
The electrodes are put into place and connected to an 18V battery. This is left to run for 6-8 hours. Alternatively, a higher power voltage pack can be used and the gel run for a much shorted time (less than 2 hours).
131
During elecrtophoresis, when should you not use a higher voltage?
Do not use a higher voltage unless the current is limited to 5mA, otherwise there is a risk of severe electric shock from the electrodes or gel.
132
In electroscopes, what will the speed of the different DNA fragments depend on?
Smaller fragments travel faster, so in a fixed period they will travel further.
133
What is done at the end of electrophoresis?
At the end of the period, the buffer solution is poured away and a dye is added to the gel. This dye adheres to the DNA and stains the fragments.
134
How can you separate proteins?
Similar to separating DNA, elecrophoresis can be used.
135
How is separating proteins with elecrophoresis different to that separating DNA fragments?
The separation of proteins is often carried out in the presence of a charges detergent such as sodium dodecyl sulfate (SDS), which equalises the surface charge on the ,molecules and allows the proteins to separate as they move through the gel according to their molar mass.
136
What does the presence of sodium dodecyl sulfate (SDS) do during elecrophorysis of proteins?
It equalises the surface charge on the molecules and allow proteins to separate as they move through the gel according to their molar mass.
137
What are proteins separated by when they undergo electrophoresis in the presence of sodium dodecly sulfate?
By molar mass.
138
Without the presence of sodium dodecyl sulfate, what will proteins be separated by during ekectrophosis?
They will be separeated by their surface charge.
139
What can protein elecrophoresis be used for?
It can be used to analyse types of haemoglobin proteins for the diagnosis of different conditions.
140
Protein elecrophoresis can be used to analyse types of haemoglobin proteins for the diagnosis of different conditions, what conditions?
Sickle cell anaemia, where the patient has haemoglobin S and not the normal haemoglobin A.
Aplastic aneamia, thalassaemia and leukemia, where the patients have higher than normal amounts of fetal haemoglobin (haemoglobin F), and lower then normal amount of haemoglobin A.
141
What is a DNA probe?
A short (50-80 nucleotides) single-stranded length of DNA that is complementary to a section of the DNA being investigated.
142
What may a DNA probe be labelled by?
A radioactive marker
A florescent marker
143
Desacribe a radioactive marker used to label a DNA probe.
A radioactive maker, 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.
144
Describe a florescent marker used to label a DNA probe.
A florescent marker that emits a colour on exposure to UV light. Fluorescent markers may also be used in automated DNA sequencing.
145
What are DNA probes useful for?
Theyre useful for locating specific DNA sequences.
146
What are some examples where DNA probes are useful in location specific DNA sequences?
.To locate specific gene needed for use in genetic engineering.
.To identify the same gene in a variety if different genomes from different species when conducting genome comparison studies.
.To identify the presence or absence of a specific allele for a particular genetic disease or that gives susceptibility to a particular condition.
147
What is a microarray?
scientists place a number of different probes on a fixed surface- this is a DNA microarray.
148
What is DNA mircoarrays used for?
Applying the DNA under investigation to the surface can reveal the presence of mutated alleles that match the fixed probes, because the sample DNA will anneal to any complementary fixed probes.
149
Before using a DNA mirocarray, what must do done to the DNA?
The DNA sample must be broken down into smaller fragments, and amplified by PCR.
150
What can a DNA mircoarray be made with?
It can be made with fixed probes, specific for certain sequences found in mutated alleles that causes genetic diseases.
151
During analysis with a DNA microarray, what is done to the reference and test samples?
The reference and test samples are labelled with fluorescent markers.
152
During analysis with a DNA microarray, why are reference and test samples labelled with fluorescent markers?
When a test subject and a reference marker both bind to a particular probe, a scan reveals fluorescence of both colours, indicating the presence of the particular sequence in the DNA test. It shows it is working.
153
What is DNA ligase?
An enzyme that catalyses the joining of sugar and phosphate groups within DNA.
154
What is elecropropogation?
A methord for introducing a vector with a novel gene into the cell; a pulse of elecricity makes the recipient vell membrane porous.
155
What is a plasmid?
small loops of DNA in prokaryotic cells.
156
What is recombiant DNA?
A composite DNA molecule created *in vitro* by **joining forgein DNA with a vector** molecule such as a plasmid.
157
What are restriction enzymes?
ENdonuclease enzymes that cleave DNA molecules at specific recognition sites.
158
Wat is a vector in gebne technology?
Anything that can carry/ insert DNA into a host organism; inculding, plasmids, viruses, certain bacteria.
159
What is genetic engineering also known as?
Recombiant DNA technology or genetic modification.
160
Why is genetic engineering also known as recombinant DNA technology?
Because it involvers combing DNA from different organisms.
161
What 4 stages as nessessaryb in genetic engineering?
* The required gene is obtained.
* A copy of the gene is placed inside a vector.
* The vector carries the gene into a recipient cell.
* The recipient expresses the novel gene.
162
What are the 4 different ways of obtaining the required gene in genetic engineering?
* Using reverse transcriptase.
* Using an automated polunucleotid synthesiser.
* PCR
* Restriction enzymes.
163
How can reverse transcriptase be used to obtain the required gene in gentic engineering?
mRNA can be obtained from cells where the gene is being expressed. An enzyme, reverse transcriptase, can then **catelyse the formation of single strand of complementary DNA (cDNA)** using the **mRNA** as a **template**. The addition of **primers and DNA polymerase** can make this cDNA into a double-stranded length of DNA, whose base sequence codes for the orginal protein.
164
How can an automated polynucleotide synthesiser be used to obtain the reqired gene in genetic engineering?
If scientsis **know the nucleotide sequence of the gene**, then the gene can be synthesised using a automated polynucleotide synthesiser.
165
What can PCR be used to obtain a required gene in gentic engineering?
If scientists **know the sequence of the gene**, they can design polymerase chain reaction primers to amplify the gene from the genomic DNA.
166
Hoe can restiction enzymes be used to obtain a required gene for genetic engineering?
A **DNA probe** can be used to locate a gene within the genome and the gene can then be cut out using restiction enzymes.
167
How is a gene placed into a vector?
* **Plasmids** can be obtained and mixed with **restriction enzymes** that will cut the plasmid as specific recognition sites.
* The cut plasmid has exposed 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. **DNA ligase** enzymes caalyse the annealing.
* A gene may be sealed into a attenuated virus that could carry it into a host cell.
168
Why does getting the vector into the recipient cell require different processes?
DNA does not easily cross the recipient cells plasma membrane, so processes can be used to aid the process.
169
What are the 5 ways that can be used to get a vector into the recipient cell in genetic engineering?
* Heat shock treatment
* Elecroporation
* Electrofusion
* Transfection
* T1 (recombiant) plasmids
170
How can heat shock treatment be used to get a vector into the recipent cell in genetic engineering?
If bacteria are subjected to alternating periods of cold and heat in the **presence of calcium chloride**, their walls and membranes will become **more porus** and allow in the recombiant vector.
171
Why does heat shock treatment get a vector into the recipient cell?
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 forgein DNA and the host membranes.
172
How can elecroporation be used to get the vector into the recipient cell in genetic engineering?
A high voltage puse is applied to the cell to disrupt the membrane.
173
What is it called to use an electric pulse to disrupt the membrane in a cell to get a vector into the recipent cell?
Elecroporation.
174
What is called when bacteria are subjected to cold and heat to get the vector into the recipient cell?
Heat shock treatment.
175
How is elecrofusion used to get the vector into the recipent cell in genetic engineering?
Electrical feilds help to intoduce DNA into the cells.
176
What is it called when electric feilds are sued to get a vector i n the the recipient cell in genetic enguneering?
Electrofusion.
177
How was T1 (recombiant) plasmids used to get the vector into the recipient cell?
T1 plasmids are inserted into the bacterium ***Agrobacterium tumefaciens***, which infects some plants and naturally inserts its genome into the host cell genomes.
178
How can you directly introduce a gene into the recipient if the plant is not susceptible to A.*tumefaciens*?
Small pieces of gold or tungesen are coated with the DNA and shot into the plant cells. This is celled a 'gene gun'.
179
What are restriction enzymes in bacteria and archaea celled?
Restriction endonucleases.
180
How does restriction nucleases protect bacteria and archeae?
They protect them from the attach by phage viruses. These enzymes cut up the foreign viral DNA, by a process called restriction, preventing the virus from making coppies of themselves.
181
How is prokaryotic DNA protected from the action of restriction endonucleases enzymes?
The prokaryote is methylated at the recognition sites, therefore pretecting itself from the action of the endonucleases.
182
How are restriction enzymes useful at molecular biology and biotechnology?
They can be used as molecular sissors.
183
What sort of ends can restriction enzymes produce?
Sticky ends or blunt ends.
184
What reaction does DNA ligase enzymes catalyse?
It catalyses condensation reactions that join the sugar groups and phosphate groups of the DNA backbone.
185
What must be obtained to make genetically modified bacteria that produce insulin?
mRNA from beta cells of islets of Langerhans in the human pancreas.
