Genome projects and gene technologies Flashcards
What is it called when a gene is cloned in a living organism?
In vivo cloning
What is cloning outside of the body called?
In vitro cloning
What are the two way of carrying out gene cloning?
In vitro and In vivo cloning
What do scientists need to use when inserting a gene into the host cell?
A vector
What does a vector do?
Carries the DNA fragment into the host cell
For bacteria, what is a commonly used vector?
Plasmid
What forms between the complementary bases of the DNA fragment and plasmid?
Hydrogen bonds
what enzyme is used to join the nucleotides of the plasmid and DNA fragment?
DNA ligase, which forms a phosphodiester bonds
What does DNA ligase do?
Catalyses the formation of a phosphodiester bond
What is the plasmid called when the DNA fragment has joined to it?
Recombinant plasmid
For In vivo cloning, do scientists use sticky ends or blunt ends?
Sticky ends
In vivo cloning use DNA fragments produced by…
Restriction endonucleases
Explain why the same restriction endonuclease must be used to produce on both the DNA fragment and the plasmid during in vivo gene cloning.
The restriction endonuclease cuts both the DNA fragment and the plasmid at the same recognition sequence.
This means that the sticky ends on both are complementary to each other.
As a result, the DNA fragment and the plasmid can slot together with hydrogen bonds between the complementary bases.
What is the sequence called where transcription starts?
Promoter
What s the sequence called that acts a de attachment site, and ends transcription?
Terminator
Does the DNA fragment contain a promoter or terminator?
No
What is a promoter sequence?
A sequence of bases that acts as a binding site for RNA polymerase and transcription factors, so it promotes transcription
What is a terminator sequence?
A sequence of bases that act asa de attachment site for RNA polymerase and transcription factors, so it terminates transcription
what stage of protein synthesis are promoters and terminators involved in?
Transcription
During In vivo cloning, what needs to be added to the plasmid alongside te DNA fragment?
A promoter and a terminator
What are two ways to make it easier for plasmids to pass a cell surface membrane?
- Add calcium ions
2.Heat shock
(rapidly increasing the temperature)
(increases spaces between adjacent phospholipids in the membrane, creating holes in the membrane for the plasmid to travel through)
What is the the process of adding a plasmid to a host cell called?
Transformation
What does transformation require?
.Heat shock
.Calcium ions
Describe and explain the process of transformation during in vivo gene cloning.
Transformation is the process in which a plasmid is added to a host cell.
It requires the addition of calcium ions to the mixture, and heat shock in order for plasmids to pass through the cell surface membranes of bacteria.
What are the issues during in vivo cloning?
.The plasmid may close up and not include the DNA fragment
.The DNA fragments might join together, and if this enters the bacteria/host cell, it will identify it as foreign, and the bacteria/host cell will be destroyed
.Some bacteria won’t take up recombinant plasmid, it will take up normal plasmid during transformation
Scientists begin in vivo gene cloning using plasmids that contain a gene for antibiotic resistance.
This antibiotic resistance gene means that any bacteria which have taken up a plasmid…
Will be resistance to the antibiotic
Bacteria which have taken up a plasmid will be resistant to a particular antibiotic.
Therefore, after transformation, when the bacteria are placed on a plate containing this antibiotic…
Only bacteria without a plasmid will die
What is the antibiotic resistance gene called?
A marker gene
To identify which bacteria have taken up a plasmid, scientists use a plasmid that contains an…
Antibiotic resistance gene, so any bacteria without a plasmid will die
The addition of a DNA fragment stops the second antibiotic resistance marker gene from functioning.
Therefore, any plasmid which has taken up the DNA fragment…
Won’t provide resistance to the second antibiotic
The bacteria are placed in a plate containing the second antibiotic.
Which of these bacteria will die?
Bacteria containing the recombinant plasmid
What does replica plating involve?
.Transferring a sample of the bacteria onto a new plate
.Comparing the two samples of the same bacteria
During in vivo gene cloning, bacteria are transferred to a plate containing an antibiotic (kanamycin).
Explain the purpose of this step.
To identify the bacteria that have taken up a plasmid.
Since the plasmid contains a resistance gene for kanamycin, bacteria without a plasmid will die when plated on kanamycin.
During in vivo gene cloning, bacteria are grown on a plate containing an antibiotic (kanamycin).
The surviving bacteria are then transferred to a plate containing a second antibiotic (ampicillin).
Explain the purpose of this step.
To identify the bacteria that have taken up the recombinant plasmid (DNA fragment) , rather than a plasmid without a DNA fragment.
The addition of the DNA fragment into the plasmid disrupts the function of the ampicillin resistance gene. This means that when plated on ampicillin, bacteria that contain the recombinant plasmid (i.e. contains the DNA fragment) will die.
The addition of a DNA fragment disturbs the fluorescence marker gene.
This means that bacteria containing the recombinant plasmid…
won’t glow
As well as antibiotic resistance genes, what do scientists also use?
Fluorescent marker gene
Enzyme marker gene
Fluorescent and enzyme marker genes provide observable properties in the bacteria.
Bacteria which have taken up the recombinant plasmid…
don’t display the observable property
Which of the following is a benefit of using a fluorescent marker gene over an antibiotic resistance marker gene?
Fluorescence is an observable property.
There is no risk of antibiotic resistance being passed onto other bacteria.
Fluorescence marker genes are more economical.
There is no need for replica plating so it is faster.
What is PCR used for?
The PCR produces lots of DNA fragments in a continuous cycle
What does PCR require to copy a DNA fragment?
.DNA polymerase
.DNA nucleotides
.Primers
How fast is the PCR compared to in vivo gene cloning?
The PCR is faster
When a Biologist carries out PCR, some contaminating DNA might enter the sample.
Does PCR multiply this contaminating DNA?
Yes
What is a DNA probe?
Singe-stranded piece of DNA with bases complementary to a specific gene sequence
What is DNA hybridisation?
DNA probe anneals/hybridises/binds/attaches to its complementary DNA sequence
When a DNA probe attaches to a complementary DNA sequence, we call this process…
DNA Hybridisation
What are DNA probes made from?
DNA fragments
Adding fluorescent dye to a DNA probe won’t make it visible under a special light.
Just as a radioactive DNA probe needs activating, the fluorescent probe must first…
bind to its complementary DNA sequence
What are the two different types of labelling probes?
Fluorescent and radioactive
What are the differences between a radioactive probe and a fluorescent probe?
Radioactive:
.Use a special scanner
.Are more dangerous
.Replace part of the probes’ molecular structure
Fluorescent:
.Use a special light
.Are safer
What do labelling methods (of probes) allow scientists to do?
To identify whether a DNA probe has binded to its complementary DNA sequence
The substance used to label a DNA probe radioactively is the radioactive form of…
Phosphorus
The substance used to label a DNA probe fluorescently is…
Fluorescent dye
What does applying heat to DNA cause?
.Hydrogen bonds between bases to break
.DNA strands to separate into two complementary strands
What are the steps of DNA hybridisation?
1.Multiply DNA using PCR
2.Heat DNA sample
3.Add DNA probes and cool mixture
4.Wash DNA sample (with ethanol) (removes any unbound DNA probes)
5.Check for possible attachment of DNA probe
How do you prevent te the DNA strands from binding to eachother?
You increase the number of DNA probes
When the DNA probe glows under the special light or scanner, this means that…
. DNA hybridisation between one of the DNA probes and a DNA sequence has happened.
. the gene being looked for is present.
What are VNTRs?
VNTRs are repetitive, non-coding DNA sequences that are directly next to eachother
VNTRs can vary in their…
number of repeats
Explain why there is a wide variation in the patterns of repeated VNTRs
.VNTRs are non-coding
.Natural selection doesn’t have an effect on the variation of number of repeats in VNTRs
.So individuals pass them onto their offspring, and they vary even more by mutations
Describe the two steps required to isolate VNTRs from a sample of DNA
.The DNA fragment is amplified using PCR
.Then, the VNTR regions and isolated using restriction endonuclease
What can genetic fingerprinting be used for?
.To determine the relatedness of animals and plants
.To determine variability within a population
Why are VNTRs used in genetic fingerprinting?
They are almost always unique to an individual
describe the steps of gel electrophoresis
What is the charge of DNA?
Negative
What length of fragment travels faster?
Shorter fragments
Why does gel electrophoresis work?
Because DNA is charged (negatively)
the more negative the charge… (gel electrophoresis)
the further it will travel down the gel
we can use gel electrophoresis to investigate sample of…
.DNA
.RNA
.Proteins
in gel electrophoresis, protein molecules can be separated by…
Length AND charge
In gel electrophoresis, molecules of DNA and RNA can be separated by…
Length (not charge, because all DNA and RNA fragments have the same negative charge)
outline the process of genetic fingerprinting
First, DNA is collected from a sample tissue.
Second, PCR is used to amplify the DNA.
Third, the VNTRs are cut out as DNA fragments using restriction enzymes.
Fourth, the VNTR DNA fragments are separated by gel electrophoresis.
Fifth, an alkali is added to the gel, to separate the VNTRs into single strands.
Then, DNA probes complementary to the VNTR sequences are added to identify the VNTRs ready for analysis.
Probes are short polynucleotide made using nucleotides that either…
a) contain radioactive phosphate
b) attached to fluorescent tags
