Pack 22 Gene Technology Flashcards
(28 cards)
Ways of making DNA fragments
Restriction endonuclease gene machine reverse transcriptase
restriction endonuclease
- there are many types of restriction endonuclease, cutting DNA at a specific sequence of bases, termed recognition sequences.
- they hydrolyse phosphodiester bonds in the sugar phosphate backbone
- these ‘cuts occur between opposite base pairs in a staggered fashion to create sticky end
sticky ends
single stranded sections of DNA that form an overhang at the end of a double stranded molecule
gene machine
- the amino acid sequence is determined
- from this, the mRNA codons for each amino acid are looked up
- the complementary DNA triplets are worked out and the gene produced
advantages of gene machine
- that any sequence of nucleotides can be produced in a very short period of time and with great accuracy
- genes would be free of introns and other coding DNA
reverse transcriptase
-the enzyme reverse transcriptase can be used to convert mRNA in a cell to cDNA
two advantages of using reverse transcriptase
- mRNA is at a high concentration within cells therefore it is easier to isolate compared to a gene
-the cDNA produced will have the non-coding introns removed. the gene will therefore to able to be expressed in prokaryotes
polymerase chain reaction (pcr)
- the temperature temperature begins at 95c. this breaks hydrogen bonds to separate the double strands of DNA
- the temperature is reduced to 55c, causing the primers to attach to complementary base sequences at the ends of the single strands
- DNA polymerase attaches and the temperature is raised to 72c. this is the optimum for this enzyme
- free nucleotides attach to the single stranded template by complementary base pairing.
- DNA polymerase makes new strands by joining the free nucleotides together with phosphodiester bonds
what happens at the end of the cycle
- the process is repeated and the dna is doubled in quantity each time
thing to calculate number after certain number of cycles
2 to the power of the number of cycles
gel electrophoresis
- DNA samples are first amplified using PCR then cut into fragments using restriction enzymes
- the fragments are placed into wells at one end of a thin slab of gel
- an electric current is passed through the gel. the DNA is attracted to the positive electrode.
- the molecules have to move through the gel, and shorted lengths of DNA move faster than larger lengths. so, the shorter the length of the DNA molecules, the further down the gel it will move in a given time.
the separated fragments on the gel cannot be seen
so must be marked
- the DNA can be stained with a coloured or fluorescent chemical
- the DNA samples can be radioactively labelled. an x-ray film is used to identify the positions of DNA fragments
what is a gene probe
single stranded, short sequence of DNA nucleotides with a complementary base sequence to the gene being screened for.
genetic screening and diagnosis
- following gel electrophoresis gene probes can be used to identify whether a particular gene, or allele is present within any of the DNA fragments
- a gene probe will bind to the target gene if it is present in a sample of DNA
- the gene probe is usually labelled with a fluorescent or radioactive tag
- the bound gene probe can the be visualised
use of a DNA gene probe in genetic screening
- PCR is used to amplity DNA containing the potential disease causing allele
- the DNA is cut into fragments using restriction enzymes
- the fragments are separated using gel electrophoresis
- DNA is made single stranded using an alkali
- the gene probe is added
- the gene probe will bind to the target allele if it is present in a sample of DNA
what does genetic fingerprinting focus on?
the differences in the non-coding
what does the non coding DNA contain and info
- VNTRs
- each individual has a unique pattern of repeats of VNTRS
- the probability of two individuals having the same VNTR pattern is very low
look in pack page 15
do it
what is vivo cloning
involves the transfer of fragments of DNA from one organism or species to another
short process for vivo cloning
1- preparing the DNA fragments
2- insertion of DNA fragment into a vector
3- transformation
4- identification of the transformed cells have successfully taken up the gene
5- growth of population of transformed cells
preparing the DNA fragment for insertion- vivo phase one
the preparation of the DNA fragments often involves the addition of extra lengths of DNA this includes:
- a promoter sequence at the start to provide a binding site for RNA polymerase
- a terminator sequence at the end that releases RNA polymerase and ends transcription
vectors for vivo cloning
-plasmid
- also viruses or liposomes
why are plasmids good
- because they can be transferred into bacteria easily
how is DNA fragments inserted-vivo phase two
1- a restriction enzyme is used to cut the gene from DNA to create sticky ends
2- the same restriction enzyme cuts the plasmid to create complementary sticky ends
3- the gene and plasmids are mixed in a test tube and they join because they were cut with the same restriction enzyme and have complementary sticky ends.
4- DNA ligase added the gene and plasmid are joined by DNA ligase; this joins the sticky ends together by forming phosphodiester bonds
5- in some plasmids the sticky ends will join themselves to re-from the original plasmids
