Gene Technology Flashcards
Why is reverse transcriptase so called?
It makes DNA from RNA (opposite to transcriptase)
What is the DNA produced by reverse transcriptase called?
cDNA (complementary DNA)
Describe the steps of making a DNA fragment using reverse transcriptase.
mRNA is used as a template
Isolated from cells using a centrifuge
Mix mRNA with free DNA nucleotides and reverse transcriptase
Result is a new DNA strand (cDNA)
Why is mRNA often better for making Replicants of genes?
Only two copies of the each target gene are in cells, however there are many mRNA copies (only some cells however, we must first locate the cell with a lot of mRNA coding for the target gene in)
Describe the steps of the polymerase chain reaction (PCR)
DNA mixture heated to 95oC to separate strands (H bonds break)
Cooled to 50-60oC so primers can bind
Mixture heated to 72oC so DNA polymerase can work
Polymerase lines up free nucleotides Along DNA strand via comp. base pairing
Two copies of the fragment are formed
Process can begin again (fragments increase exponentially)
What is the reaction mixture set up for PCR?
DNA sample
DNA polymerase
Free nucleotides
Primers
What are primers and why are they needed in Replicating genes?
Short pieces of DNA that are complimentary to the bases at the start of the fragment you want
Signals where to start the replication (DNA polymerase binds here)
What is a palindromic sequence?
Sections of DNA that consist of anti parallel base pairs
What is restriction endonuclease?
An enzyme that recognises and cuts DNA at specific palindromic base sequences.
How do restriction endonuclease enzymes work?
They hydrolyse the DNA (or digest it) as the shape of the recognition site (palindromic sequence) is complimentary to the enzymes active site)
Describe how you can produce a DNA fragment using restriction endonuclease?
Palindromic sequences are at either side of the fragment / target gene you want
Restriction endonuclease enzymes cut the DNA at theses points.
This can lead to sticky ends that can bind to other DNA fragments with comp base pairing.
What is in vivo gene cloning?
Gene copies are made within a living organism. As the organism grows and replicates it’s DNA multiple replicants of the gene are made
What is in vitro gene cloning?
Where the gene copies are made outside of a living organism I.e the PCR
What is recombinant DNA?
The name for DNA formed by joining DNA together from different sources
What are vectors?
Something used to transfer DNA into a cell (virus or plasmid)
Describe the steps of making recombinant DNA
Fragment of DNA to be inserted is isolated via restriction enzymes (restriction endonuclease)
Vector DNA is isolated
Vector DNA is cut using the same restriction enzyme used to isolate the DNA fragment containing target gene so there sticky ends are complimentary
Vector and DNA fragment are mixed together with DNA ligase which joins the fragments together (by their sticky ends) - this process is called ligation
Combination of new bases is the recombinant DNA
What is the function of DNA ligase?
To join together sections of DNA (e.g sticky ends in making recombinant DNA)
What enzymes are involved in making recombinant DNA?
Restriction enzymes (for both vector DNA and isolating target gene) DNA ligase
What are transformed cells?
They are cells that take up the vector with the recombinant DNA so that the cell contains the gene you want.
Host cells that take up the vectors containing the gene you want are said to be transformed
How are plasmid vectors taken up by bacteria?
Place bacteria in Ice cold chloride solution so their cell walls become more permeable.
Plasmids added to mixture and then it is heat shocked to around 42oC which encourages the cells to take up the plasmids.
What is the problem with transforming cells?
The host cells may not necessarily take up the vector DNA containing the gene of interest. It is all due to chance
How can we identify transformed cells?
Marker genes inserted into vector DNA with gene of interest
Host cells grown on agar so they divide
Marker gene can be antibiotic resistance or fluorescent gene
Only transformed cells with survive or fluoresce.
What is genetic engineering?
The manipulation of an organisms DNA. Also known as recombinant DNA technology. Organisms are called transformed organisms - they contain the recombinant DNA
What parts of DNA are used in genetic fingerprinting?
Parts of the genome that do not code for polypeptides (introns). These consist of repetitive, non-coding base sequences
These are called minisatellite regions
Why are minisatellite regions used in genetic fingerprinting?
Theses regions vary greatly between individuals and so are unique to a particular organism
And so they can be compared
(One person may have a section that repeats 4 times, another person 17 times)
Describe the steps involved in genetic fingerprinting
Collect sample of DNA to be analysed (from tissue sample)
PCR used to make many copies of DNA
Primers bind to either side of the repeats to keep their original length
Restriction enzymes cut the DNA to isolate the repeating regions
Separate the fragments by gel electrophoresis
Separate according to length (smaller move further and faster)
Compare to a DNA ladder
Apply probe (fluorescent)
Bands of DNA show up under UV light
Describe how gel electrophoresis works.
DNA fragments placed into a well in a Slab of gel and covered in buffer solution that conducts electricity
Current passed through the gel
DNA fragments separate according to length
Shorter fragments move faster and further
DNA fragments negatively charged so travel towards positive electrode
What is the importance of a DNA ladder in gel electrophoresis?
Known lengths of DNA fragments so that fragments of unknown length we are testing for can be worked out what size they are by comparison
How can genetic fingerprinting help identify fathers of children?
Bands that do not come from the mothers DNA, will come from the DNA of the father
See how closely the bands match up
How can genetic fingerprinting help increase genetic variation in terms of breeding animals?
Pick animals with the most dissimilar bands of DNA to breed together.
This will increase genetics variability and reduce the risk of genetic disorders
What are DNA probes?
Short single strands of DNA that have a specific base sequence complimentary to the base sequence of a target gene and so will hybridise to them.
Probes can have fluorescent or radioactive tags so they show up under uv light / x rays
What is reverse transcriptase?
An enzyme that makes DNA from RNA.
Describe the steps involved in locating genes using probes
Sample of DNA is digested into fragments using restriction enzymes and then separated using gel electrophoresis
Separated fragments are transferred to nylon membrane and incubated with a DNA probe. If gene present, the probe will bind
Use detection methods of finding the probe
Describe how a restriction map is formed
The DNA is labelled and cut into fragments by DIFFERENT restriction endonuclease enzymes
Separated by gel electrophoresis
The lengths of the fragments produced are used to determine the relative locations of cut sites
What is a restriction map?
A diagram of the DNA showing different cut sites of restriction endonuclease enzymes
Describe the steps in DNA base sequencing
A mixture is set up (DNA, polymerase, primer, free nucleotides, fluorescently labelled modified base.)
Tube undergoes PCR
The strands produced are of different length depending on where the modified base is added.
Fragments are separated out by gel electrophoresis
This method is done with each of the four bases.
Under uv light u can see all the fragments, each band will be one base longer than the next.
When base sequencing, the DNA undergoes PCR but the strands will be of different length due to the modified bases. What will each of the strand end with?
The modified base - it terminates PCR (it could be any of the modified base - T A C G - depending which was added)
What does the reaction mixture composed of for DNA base sequencing?
Single stranded DNA template to sequence DNA polymerase Lots of DNA primer Free nucleotides Modified nucleotides
What is the primer used for in DNA base sequencing?
To begin the process of PCR - this is where the process begins on each strand. - they are complimentary to the bases at the start of the fragment you want
How can restriction mapping, sequencing and DNA probes help when it comes to mutations?
Genetic disorders are caused by mutations
If you know the sequence a gene should be and what the mutated versions are (via sequencing) you can screen people for genetic disorders
- early diagnosis
- help determine what cause of treatment
How do you make a probe to screen for a particular gene?
The gene of interest is sequenced.
PCR is then used to produce many copies of the gene - these are the probes
How can you screen for a single gene?
The probe is labelled to look for a single gene in a sample of DNA (will only have complementary base paring to one gene)
How can you screen for multiple genes using probes?
Probe used as part of a DNA microarray
This screens for lots of genes at the same time
Sample of labeled DNA is washed cover the microarray.
If ant DNA sequences match any of the probes, it will stick to the array
Unstuck DNA is washed away
Visualise microarray in UV light
Any labelled DNA attached to the array will show up - the person has that particular gene
What is genetic counselling?
Advising patients and their relatives about genetic disorders
Involves advising about screening
Advice on treatment and preventing if positive (carrier etc.)
What is a microarray and what is it used for?
A glass slide with microscopic spots of different DNA probes attached to it in rows
It is used to screen DNA for multiple genes using many different probes
What is gene therapy?
Gene therapy involves altering the defective (mutated) genes inside body cells to treat genetic disorders and cancers
How you carry out gene therapy to treat a disorder depends on what? (To do with the gene)
Whether the disorder is caused by two recessive alleles or a dominated allele
How is gene therapy used to treat a disorder caused by two recessive alleles?
Adding a working dominant allele to make up for the recessive alleles
-supplementing the faulty ones
How is gene therapy used when the disorder is caused by a dominant allele?
Inserting DNA in the middle of the gene so it doesn’t work
- silencing the gene
Replacing it with a functioning gene?
What are the two types of gene therapy?
Somatic gene therapy
Gene line therapy
What does somatic therapy involve?
Altering alleles in body cells, especially ones affected by the disorder.
What does gene line therapy?
Altering alleles in sex cells
Why may a person treated by somatic gene therapy still pass on the disorder onto their children?
Somatic gene therapy does not affect sex cells - the new or altered gene is not passed on to the offspring
Will the children of someone treated by gene line therapy have the genetic disorder?
No, gene line therapy involves altering the alleles in the sex cells.
This means every cell of the offspring will by affected by the gene line therapy and so will not carry the genes for the genetic disorder
What are vectors used for in gene therapy?
To get the new / altered allele into the cells
Give two examples of vectors that can be used in gene therapy?
Viruses
Liposomes
What is a liposome?
A small lipid sphere
How is a liposome used in gene therapy? How does it carry new alleles into body cells?
Insert the gene into a plasmid,
Wrap it in a liposome
These fuse with the phospholipid belayer of cells and release the DNA into the cell.
Hopefully it moves into the nucleus and is expressed
How is a virus used in gene therapy? How does it get the new / altered gene into body cells?
Take the genetic material of the virus out
Insert the material you want
The virus carries out it’s function and inserts the DNA directly into target cells
Where hopefully it will be expressed
Which cells will somatic gene therapy target when treating CF?
Epithelial cells in the lining of the lungs
What are the advantages and disadvantages of PCR?
Adv
- fast process
- produces many copies
- DNA produced not modified
- only replicates fragment of interest
- don’t have to isolate the fragment from host DNA of cells
Disadv
- only replicates a small DNA fragment
- doesn’t produce modified DNA proteins or mRNA if you want it
- can be expensive
What are the advantages and disadvantages of in vivo cloning?
Adv
- produces RNA protein etc. as well as it’s done in a living cell
- can produce modified DNA
- large fragments of DNA can be cloned
- relatively cheap
Disadv
- fragment has to be isolated from other cell components
- produces mRNA, protein etc.
- slow process
What are the benefits of transformed organisms? (Via genetic engineering)
- crops can produce better yields, more nutritious, withstand climate better
- enzymes used in industrial processes can be produced from transformed animals in large quantities for less money
- medicine and vaccines are produced from transformed organisms using recombinant DNA technology
What are the concerns of transformed organisms? (Via genetic engineering)
- farmers may only plant one type of crop making them vulnerable to diseases and reducing biodiversity
- super weeds
- no choice about eating GM food and crops
- technology used unethically - designer babies
- large biotechnology companies becoming more powerful forcing smaller companies to close
What are some uses for genetic fingerprinting?
- finding out relationships between people (fathers)
- increasing genetic variation in breeding animals
- determining genetic variation within a population
- forensic science
- medical diagnosis
What are the advantages and disadvantages of gene therapy?
Adv
- prolong lives of people with life-threatening disorders
- give people better quality of life
- gene line therapy allows people to have children without disease
- decrease number of people with genetic disorders
Disadv
- body could identify vectors as foreign and start an immune response
- allele inserted into the wrong place in DNA causing cancers
- inserted allele could be over expressed
- patient may have to go under multiple treatments
- difficult to get the allele in certain body cells
