Gene Technology Flashcards
What is recombinant DNA
Describes how new combinations of genetic material are produced by artificially copying a piece of DNA from the donor and placing it in the recipient. Produces genetically modified organisms.
What are restriction endonucleases
Enzymes which cut the DNA at specific base sequences.
-Each restriction endonuclease recognises a specific base sequence
-These enzymes are used by bacteria to defend themselves against bacteriophages.
-they restrict the growth of invading viruses by chopping the bcteriophage DNA into smaller non-infectious fragments.
-These enzymes cut the DNA double strand by a hydrolysis reaction- producing blunt or sticky ends
What are blunt ends
-cut the DNA strnads at positions directly opposite one another, giving blunt ends to the fragments
What are sticky ends?
a restriction enzyme that makes double stranded cuts between certain nucleotides on either side of the DNA strand. The cuts are staggered
- and produces single stranded regions called sticky ends
Which are more useful blunt ends or sticky ends?
sticky ends are more useful to genetic engineers because they can form base pairs with other complementary sticky ended DNA molecules.
What happends when two DNA fragments are mixed?
-The complementary bases of their ‘sticky ends’ will be attracted to one another- form HYDROGEN bonds.
-DNA Ligase seals fragments of DNA
What is reverse transcriptase
-retroviruses store genetic information in the form of RNA- they use enzymes to make DNA from RNA- enzyme involved is reverse transcriptase
-reverse of transcription
-enables a single chain of DNA to be made from the corresponding mRNA molecule- called cDNA.
Explain the technique used to make cDNA from mRNA
- ISOLATING AND EXTRACTING THE MRNA- analysing cells in which there are a lot of relevant mrna- gene is active
- Reverse transcriptase- used to make a single strand of DNA (cDNA) using the mRNA as a template and following normal base pairing rules
- enzyme DNA POLYMERASE used to make the DOUBLE STRANDED DNA (gene) from the single strand of DNA
- Tjis produces the required gene needed to code for the protein
What is a DNA probe?
A DNA probe is used to LOCATE A PARTICULAR NUCLEOTIDE SEQUENCE OR GENE on a DNA molecule
-consists of a SHORT SINGLE STRAND OF DNA that contains the known sequence of bases
-labelled either:
1. RADIOACTIVE- detected by X- ray film
2. FLUORESCENT- detected by UV light
-THE BASES IN THE DNA PROBE HYBRIDISE WITH THE COMPLEMENTARY BASES ON THE DONOR DNA, REVEALING THE POSITION OF THE GENE
Once the gene is located on the DNA probe, how can it be extracted
-Target DNA is hydrolysed by RESTRICTION ENDONUCLEASES and DNA fragments are separated by GEL ELECTROPHORESIS
-DNA sections are transferred to a NYLON MEMBRANE and the FLUORESCENTLY DNA PROBE added. Any DNA probes that have not attached are washed off as they would still show up. If the nylon membrane is exposed to UV light, the DNA Probe, and target sequence will appear as a fluorescent band
What is gel electrophoresis
-SEPARATES THE DNA SECTIONS BASED ON SIZE (LENGTH), with shorter sections travelling further through the gel
-uses electricity
Why wash off DNA probes that have not attached to the target sequence
-fluorescently labelled probes will show up whether they have hybridised with their target sequence or not, therefore would give an inaccurate result
How does gel electrophoresis separate the DNA sections
-BASIS OF SIZE (length)
-shorter sections of DNA travel FURTHER (and faster) through the gel
-SHORTEST sections closest- POSITIVE ELECTRODE (ANODE)
-longest sections closest to the NEGATIVE ELECTRODE (CATHODE)
When using DNA probes, explain why it is necessary that the target DNA is separated into single strands?
-exposed bases are necessary for binding
-if double stranded, bases are not exposed
What is PCR
Polymerase Chain Reaction- a technique used to amplify a sample of DNA
What are the steps involved in PCR
- Heat- 95 degrees- break hydrogen bonds and separate the two strands
- Cool and add primers- Primers anneal with complementary bases in the part of the DNA strand selected, nucleotides and DNA polymerase
-DNA is cooled to 40-60 degrees- allow primers to anneal to their complementary sequences on the separated DNA strands - Heat- 70 degrees- allow DNA Polymerase enzyme to extend the primers and complete the replication.
- Each original DNA molecule has now been replicated to form two molecules- cycle with the no. of DNA molecules doubling each time
What is the purpose of the primers
-stop the two DNA strands reannealing
-‘bracket’ the section of DNA to be copied
Why is DNA Polymerase used
DNA Polymerase is thermostable and therefore not denatured by high temperatures
-no extra treatment required to replace at the end of the cycle
- faster and more cost efficient
What are the possible uses of PCR
-forensic medicine
-Copy DNA from extinct organisms to identify relationships with living species
-Genetic screening and research involving genetic diseases
-resolve paternity disputes
What is one problem with PCR
Having a pure enough sample of DNA to start with- any contaminant DNA will also be amplified
