control of gene expression Flashcards
what is a gene mutation defined as
any change to one or more nucleotide bases or rearrangement of bases
what can rate of cell division depend upon
environment, but also genes
what could happen to cell division if gene mutation occurs
the rate of cell division is controlled by genes, if a mutation occurs in those genes then uncontrolled cell divison can occur. CANCER . when these cells keep divided they layer on top of each other forming a tumour
what are proto-oncogenes
genes that can cause cancer when they are turned on
what are tumour suppressor c
what are three methods to produce DNA fragments
conversion of mRNA to cDNA using reverse transcriptase
- using restriction endonucleases to cut fragments containing the desire gene from DNA
- creating the gene in a gene machine based on a known protein structure
describe the method to produce DNA fragments using reverse transcriptase
- the beta cells in the islets of langerhan are specialised to make insulin they therefore make a lot of mrna that codes for insulin
- this mrna will act as a template on which a single stranded complementary copy of DNA is made cDNA is formewd using reverse transcriptase
- cDNA is then isolated via hydrolysis of the mRNA using an enzyme
- double stranded dna is formed on the template of the cDNA using DNA polyermase
this is a copy of the gene
describe the mechanism of using a restriction endonucleases
- restriction endonucleases cute up DNA
- these naturally occur in bacteria as defence mechanisms
- each enzyme recognises a specific DNA sequence ( recognition site) which it is complementary to
- the DNA is cut by breaking phosphodiester bonds at the recognition site
- this will leave sticky ends whcih can easily add recombinant DNA
how does the gene machine work
the primary structure of the DNA is examined to identify the amino acid sequence then mRNA and dna can be worked out
- the dna sequence can be entered in a computer which checks for biosafety and biosecurity
- the computer can then create small section of overlapping single stranded nucleotides called oligonucleotides
the oligonucleotides can then be joined to create DNA for the entire gene - using sticky ends the gene can then be inserted into a plasmid which acts as a vector for the gene allowing it to be stored, cloned or transferred in the future
what is an oligonucleotide
small section of overlapping singled stranded nucleotides
what is an advantage and disadnavntage of the reverse transcription metjod of making dna fragment
MANY NAYURALLY OCCURING MRNA ##
but this process is more difficult
what are the adv and dadv of using endonucleases to form dna fragments
produces sticky ends so recombinant DNA can attach easily
produces introns - bacteria should not have introns
what are advantages and disadvantages of gene machine
specific so that introns can be removed, quick
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what is a palindrome
where two sequenecs are opposites of one another
what does in vivo mean
tranferring the fragments to a host cell using a vector
what does in vitro mean
uses the polymerase chain reaction
what is a recognition site
the sequences of DNA that are cut by restriction endonucleases
what does DNA ligase do
joins complementary sticky ends
REMEMBER
USE THE SAME RESTRICTION ENDONUCLEASE TO BE ABLE TO COMBINE COMPLEMENTARY STICKY ENDS TOGTHER at plasmid and at dna
what does the preparation of a DNA fragment involve
addition of extra lengths of DNA
what is a promoter region
the binding site of RNA polymerase whcih will allow transcription to occur
what is terminator region
where another region of DNA releases RNA polymerase and stops transcription of the first little strand
obvs there will need to be another terminator region at the end of DNA to stop it all
briefly describe gene transfer
- isolation of DNA fragments that have a gene for a desired protein
- insertion of DNA fragment into a vector
- transfer the DNA into a suitable host cell
- identify host cells that ahve successfully taken up the gene by use of gene markers
- growth by cloning of the population of host cells
what can be used as gene markers
fluorescence’s causes cells to glow under UV light
- bacterial cells which have not taken up the plasmid will not be antibiotic resistant
what is a vector
a carrying unit
what are vectors used for
used to transport DNA into the host cell
where do restriction endonucleases usually break in the plasmid
the gene for antibiotic resistance
describe the transformation process
plasmids and bacterial cells are mixed together in a medium containing calcium ions. the calcium ions and changes more permeable allowing the plasmids to pass through the cell surface membrane – however not all the bacteria will posses the DNA of the desired gene
what are some reason that the desired gene may not be taken up by bacterial cells
-some plasmids may have closed without taking in the DNA fragment
- sometimes the DNA fragments join together to form its own plasmid
what can PCR be described as
in vitro method of amplification
what is the purpose of PCR
to produce large quantities of of DNA or RNA fragments
what equipment is needed for PCR
- thermocycler
- DNA fragment that intends to be amplified
- DNA polymerase ( taq polymerase)
- primers
DNA nucleotides
describe the process of PCR
- PCR mixture is heated to high temperature 95 degrees. this will break hydrogen bonds to separate the DNA strands
- reduce the temperature ( 55 degrees ) so primers can anneal to DNA
- heat to higher temp (72 degrees ) which is optimum temp for DNA polymerase
- An enzyme adds adjacent nucleotides that will form phosphodiester bonds.
this will make 2 stranded DNA
repeat this cycle 25-25 times
how many times should the PCR cycle be repeated
25-35
what does heating the PCR mixture to 95 degrees do
breaks hydrogen bonds between DNA strand
NO HYDROLYSIS
what is a primer
a short single stranded
DNA sequence
what is the purpose of the PCR mixture being cooled
so primers can anneal to complementary bases at the end of DNA fragemnts
what is the purpose of PCR
DNA amplification
describe genetic fingerprinting
DNA is extracted by fractionation and ultracentrifugation
( if the sample is super small the PCR is used to amplify DNA sample)
- restriction endonucleases are added to cut the DNA into fragments
- these samples are added to wells in agar gel.
- the gel is placed in a buffer liquid with an electrical voltage applied
- DNA is negatively charged therefore the DNA samples move through the gel towards the positive end of the gel
- -the agar gel creates resistance for the moving DNA, and smaller pieces of DNA can move faster and further along the gel - this is how different lengths of DNA are separated (VNTRs) ahhhhhhhhh
that bit was gel electrophoresis
an alkaline is added to separate the double strands of DNA
Why is an alkaline added after gel electrophoresis
to separate the double strands of DNA
what can genetic fingerprinting be used for
to analyse genetic relationships and genetic variability within a population
what is a VNTR
variable number tandem repeats -
found in the non-coding region of DNA.
between individuals contain variable lengths of repeating DNA
how do you analyse genetic fingerprinting
add a DNA probe which is a single stranded piece of DNA complementary to VNTRs
- these probes are radioactively or fluorcescently labelled
- the probes are mixed in with the agar gel - they hybridise
- the agr gel will shrink and crack as it dries. the VNTRs and DNA probes are transferred to a nylon sheet. this nylon sheet is exposed to X-rays to visualise the position of the radioactive probes or UV is a fluorescent probe was used
DNA bands are then compared to identify geentic relationships
