Biotechnology Flashcards
Ligation Enzyme
DNA ligase is an enzyme that is able to join/recombine separate pieces of DNA
Can produce one longer piece of DNA or a circular molecule of DNA if the fragments have complimentary sticky ends at both ends.
Recombinant DNA technology
Involves the introduction into cells o f DNA that is foreign to the organism or has been modified in some way
Transgenic Organism: are those whose genome has been altered by the transfer of a gene or genes from another organism
Joining of Cut DNA into vector
Plasmid DNS is cut at one point using a restriction enzyme that creates sticky ends
DNA of passenger fragments Is cut with same enzyme
DNA fragments and the plasmid are mixed together and their sticky ends pair
DNA ligase is used to make the joins permanent
Plasmid is then reintroduced into the bacterial cell
Gel electrophoresis
DNA fragments can be sorted using this technique
Sorted by their lengths
Dissolved DNA fragments are placed in wells at one end of the gel
gel is then exposed to electric field (negative pole at starting end)
DNA fragments are negatively charged and so move to positive end
Shorter fragments move faster than larger ones and therefore make it further through the gel
End result is a series of parallel bands of DNA fragments at differing distances down the gel
Size of unknown bands is identified by comparing to distance moved against fragments of known size
Polymerase Chain Reaction
Segments of DNA is artificially multiplied through a series of repeated cycles
Step 1: DENATURATION
Target DNA is obtained and heated to 98 degrees. DNA denatures due to heat and the strands separate into two strands
Step 2: ANNEALING
sample cooled to approx 60 degrees
Primers (short segments of artificial single stranded DNA) are added (annealed) have complimentary bases to the start and ends of the target DNA. Primera act as starting sequence fro extension
Step 3: EXTENSION 72degrees DNA polymerase (heat resistant) reads sequence starting from the Primers and add free nucleotides creating a complimentary strand
Not until the third cycle that you get a copy of the target DNA only. After this cycle many many copies of the target DNA are produced.
Gene Probes
Pieces of DNA labelled with radioactive isotope or fluorescent marker that will bind to specific sequences of bases in another DNA molecule.
Use to detect presence of a particular sequence of bases
Abnormal genes are identified by mixing the probe and a piece of DNA
Prove seeks out complimentary bSe sequence and binds to it -> exactly locating where the target gene is located.
If gene is normal probe will bind
If abnormal gene the probe won’t bind = gap in DNA being tested
Sanger Sequencing
Dideoxynucleotides terminate DNA elongation at each base
Produces DNA fragments of variable length each piece ending with one of four ddNTPs
Labelled by a colour marker so it can be visualised when ran through electrophoresis gel. Seperate it by size
Smallest fragments are further down the cell
Complimentary sequence to the original strand is read from the bottom up.
Short tandem Repeats
Are short sequences of Nucleotides found in non coding regions of our DNA on chromosomes that are repeated a number of times (this number differs between Individuals)
Eg at a particular position on chromosome 7 the word GATA is repeated between 6 and 15 times. This number varies from person to person.
2 chromosomes from each pair, one from your father and one from your mother correspond to the same sequence of genes. You’re an individual will inherit two different number of repeats eg 5 repeats from dad and 6 from mum therefore there profile for that location is 5,6.
Australia uses STRs at 9 places.
Making proteins
Vector used is a bacteria or yeast. These will transcribe and translate the gene into a protein, which can then be harvested from the cell
Eg Insulin in E.Coli and yeast
Once gene is inserted into host cell they are subjected to rapid culture
Products of gene then produced by host cells gene expression.
When the cell expresses the new protein it either is diffuses out of the cell (secreted) or is directly obtained from cell
To deliver a gene
(Where the interest is not based on obtaining the protein produced) requires a virus or liposome to be used.
If it is a virus: used to infect the cells of a patient with its own newly recombined DNA.
Retrovirus will splice its own RNA based code permanently into the DNA of the cells they affect.
Restriction Enzymes
Are sequence specific. They recognise and bind to specific DNA sequences
Once bound to their recognition sequence the enzymes cut the sugar-phosphate backbones of the DNA strands.
Can produce a straight cut: results in clean break across the two strands to produce a blunt end
Staggered Cut: results in fragments with sticky ends. A sticky end is a stretch of unpaired nucleotides (an overhang)
Overhangs from one fragment can be paired with any other fragment of DNA with corresponding sequence
Liposome
Ball of phospholipids
Easily transferred to any site on the body
Delivery directly occurs into the intended cells.
Avoids undesirable effects eg from virus.
Uses of recombinant technology
Making proteins eg Factor VII
Hormones eh Insulin or human Growth Hormone
vaccines eg Flu Vaccine
