5: DNA Technology Flashcards
Describe genetic fingerprinting
DNA obtained
PCR makes many copies of areas that contain repeated sequences, primers are used that bind to either side of these repeats so the whole repeat is amplified
Restriction endonucleases used to cut DNA
Electrophoresis separates strands according to size
DNA fragments are transferred from the gel to nylon membrane
Radioactive DNA probes are added
Developed using X-ray film (autoradiography)
How do you improve accuracy of genetic fingerprints used in paternity tests?
Compare are higher number of places on the genome
Uses of genetic fingerprinting x3
Determining genetic relationship
Determining genetic variability within a population
Forensic science
When is genetic fingerprinting useful in genetic diagnosis?
When the specific mutation isn’t know or where several mutations could have caused the disorder, because it identifies a broader, altered genetic pattern
Why are two people unlikely to have the same genetic fingerprint
The probability of two individuals having the same number of sequence repeats at each place they’re found is very low
Role of short tandem repeats in genetic fingerprinting
Some junk DNA consists of repetitive, non coding base sequences
The number of times they repeat differs in each individual
The number of times the sequence is repeated at different places in their genome can be compared
Probability of two individuals having the same number of sequence at each place is very low
Role of reverse transcriptase
Why is it useful?
Makes DNA from RNA
Useful as many cells only contain two copies of each gene, making it difficult to obtain the target gene. But they can contain many mRNA molecules
What is meant by a palindromic sequence and why are they useful?
Sections of DNA that consist of antiparallel base pairs (base pairs that read the same in the opposite direction)
Restriction endonucleases recognise these sequences and cut the DNA at these places.
Sometimes the cut leaves sticky ends and sometimes blunt ends
Name the components of the PCR reaction mixture and explain their function
The DNA sample - may be a section cut by restriction endonucleases
Free nucleotides - which will form the copied DNA
Primers - short pieces of DNA that are complementary to the bases at the start of the fragment you want, allow DNA polymerase to copy DNA
DNA polymerase - enzyme that binds nucleotides with phosophodiester bonds
Describe the 5 steps of PCR
- Heat to 95 to break the H bonds between the two strands of DNA
- Cool 55 to allow primers to anneal to DNA
- Heated to 72 as this is DNA polymerases optimum temperature
- DNA polymerase lines up free nucleotides alongside each template strand. Specific base paring means new complementary strands are formed
- The cycle is repeated
- The amount of DNA doubles each cycle
Name 3 stages of in Vivo cloning
- Gene inserted into a vector
- Vector transfers the gene into host cells
- Transformed host cells are identified
Describe how a gene is inserted into a vector
The vector DNA is cut open using the same restriction endonuclease that was used to isolate the DNA fragment containing the target gene. The sticky ends of the vector are complementary to the sticky ends of the gene.
The vector DNA and DNA fragment are mixed together with DNA ligase. This joins the sticky ends together.
The new combination of bases in the DNA is called recombinant DNA
Describe how the vector transfers the gene into host cells
a) using a plasmid vector
b) using a bacteriophage vector
a) Host bacterial cells are placed into ice-cold calcium chloride to make their cell walls more permeable. The plasmids are added and the mixture is heat shocked, which encourages the cells to take in the plasmids.
b) The bacteriophage will infect the host bacterium by injecting DNA into it. The phage DNA then integrates into the bacterial DNA
During step 2 of in vivo cloning, not all the bacterial cells will posses the DNA fragments. Suggest two reasons for this.
- only a few bacterial cells take up the plasmids when the two are mixed together (1%)
- some plasmids will have closed up again without incorporating the DNA fragment
Describe how transformed cells are identified
Marker genes can be inserted into vectors at the same time as the gene to be cloned.
Marker genes can code for fluorescence, antibiotic resistance or an enzyme
Advantages of in vivo cloning, compared to in vitro x6
- Useful when we wish to introduce a gene into another organism e.g. gene therapy
- It involves almost no risk on contamination, sticky ends mean contaminant DNA will not be taken up, in vitro requires a very pure sample
- It is very accurate, as mutations are rare
- It makes copies of very specific genes
- It produces transformed bacteria that can be used to produce large quantities of gene products
- can produce modified DNA
Advantages of in vitro cloning, compared to in vitro x2
- It is extremely rapid, take hours rather than days
- It does not require living cells, no complex culturing techniques
What is a DNA probe?
short strands of ssDNA, specific base sequence that’s complementary to the gene you’re looking for. The probe also has a label attached, so that it can be detected
How are DNA probes used to find locate a gene?
Sample of DNA is digested into fragments using restriction endonucleases and separated using electrophoresis
The fragments are then transferred to a nylon membrane and incubated with the fluorescently labelled DNA probe. If the gene is present, the DNA probe with hybridise to it. the membrane is then exposed to UV light and if the gene is present there will be a fluorescent band.
How can the base sequence of a gene be determined by restriction mapping?
Different restriction enzymes are used to cut labelled DNA into fragments
The DNA fragments are separated by electrophoresis
The size of the fragments produced is used to determine the relative locations of cut sites
a restriction map of the orgincal DNA is made - a diagram of the piece of DNA showing the different cut sites, and so where the recognition sites of the restriction enzymes are found
State the components of the 4 test tubes in the Sanger method and explain their function
many ssDNA fragments of the DNA to be sequenced
a mixture of nucleotides, ATCG,
a small quantity of one of the four terminator nucleotides
a labelled primer to start the process of DNA synthesis
DNA polymerase to catalyse DNA synthesis
Describe the Sanger method and explain how it works
Binding of nuecleotides is random, so the addition of a normal or terminator nucleotide is equally likely
DNA synthesis may be terminated at any point so the DNA fragments in each test tube will be of varying lengths
All will end in the same base and can be identified because the primer is attached at the other end.
The fragments in each test tube are separated using gel electrophoresis and visualised under UV light
The complementary base sequence can be read from the gel.
The smallest fragment is at the bottom, each band after this represents one more base added.
So by reading from the bottom to the top, you can build up the DNA sequence one base at a time
In the sanger method which way to you read the complementary DNA base sequence
From the bottom to the top
Difference between germ line and somatic cell therapy
Germ line: replacing or supplementing the defective gene in the fertilised egg. this ensures that all the cells of an organism will develop normally
Somatic cell: Targets just the affected tissues, the additional gene is therefore not resent in sex cells, and so is not passed on to future generations. Treatment needs to be repeated periodically as cells are continually dying and being replaced. Long term aim is to target stem cells that give rise to mature tissues, effective in long term
