topic 8 - gene expression 2.0 Flashcards
why is determining the genome of simpler organisms good for the development of vaccines
-allows assignment of the proteins to each gene more easily because there is less non coding DNA
-this allows you to identify the antigens on the surfaces of the virus
what is recombinant DNA
combining the DNA of two different species
how can DNA fragments be made using reverse transcriptase
-mRNA is isolated from the cells
-the mRNA is mixed with free DNA nucleotides and the reverse transcriptase enzyme
-the reverse transcriptase enzyme use the mRNA as a template to synthesise a new strand of complimentary DNA called cDNA
-this only produces a single stranded cDNA so DNA polymerase is used to make it doubel stranded
how can DNA fragments be made using resritiction endonuclease enzymes
-restriction endonucleases cut DNA at specific sites called recognition site
-the recognition sites are usually palindromic meaning the DNA sequences consist of base pairs that read in the opposite directions
-the restriction endonuclease makes staggered cut that will exposed inpaired bases at each end of the fragment
how can DNA fragments be made using the gene machine
-the sequence of bases is determines
-the triplets are worked out and fed into a computer
-the computer will design short sections of DNA called oligonucleotides which are assembled into the desired gene
what is in vivo cloning
-the DNA fragment is inserted into a vector DNA
-a vector is something that it used to transfer DNA into a cell
describe the process of in vivo cloning
-a promoter region is added to the vector at the start of the fragment as this is the binding site for RNA polymerase
-a terminator region is added to the end of the gene to cause the RNA polymerase to detach and stop transcription
-the plasmid is cut open using the same restriction endonucleases that was used to isolate the DNA fragment containing the target gene
-this ensures the sticky ends of the vector dna fragment and the plasmid are complimentary to each other
-the vector DNA and DNA fragment are mixed together with DNA ligase which joines the sticky ends together to form recombinant DNA
-in transformation the vector needs to be inserted into the host cell where the gene in the DNA fragment will be expressed to create a protein
-to do this the membrane of the host cell must be persuaded to take the plasmid vector by placing it in calcium chloride solution and heat shocking it
what is a marker gene
-it is a gene that is transferred with the desire gene to enable scientists to identify if the cells have been successfully altered
-the marker gene can code for antibiotic resistance so that only transformed cells growing on an agar plate with a specific antibiotic can grow
what is PCR
the polymerase chain reaction is used to make millions of copies of a fragment of DNA in just a few hours
describe the process of PCR
-temperature is increased to 95 degrees to break hydrogen bonds and unwind DNA
-the temperature is decreased to 55 degrees so primers can attach
-DNA polymerase attaches the complementary free nucleotides to exposed bases through specific base pairing
-this forms a new strand which aligns next to the template
what are DNA probes
-short single strands of DNA which are labelles radioactively or fluorescently so they can be identified
-they are used to locate specific alleles of genes and to screen patients for a heritable condition, drug response or health risk
-a sample of patient dna is taken and heated to kake it single stranded
-the sample is mixed with DNA probes that been created to be complimentary
-if the patient has the allele their DNA will bind to the probe which can be identified by using x ray or uv light
what are vntrs
-variable number tandem sequence are base sequences that do not code for proteins and repeat next to each other over and over
-the number of times a sequence is repeated at different places in their genome can be compared between individuals which is called genetic fingerprinting
recall genetic fingerprinting
- collect the smallest sample of DNA
-pcr is used to make copies of the areas of DNA that contain vntrs
-a fluorescent tag is added to all DNA fragments
-the DNA samples are loaded into small wells in agar gel
-the gel is placed in buffer liquid with the electrical voltage applied
-DNA is negatively charges so the DNA samples move through the gel towards the positive end of the gel
-smaller DNA fragments move faster through the gel so the DNA fragment separate according to size
-gel is then immerse in alkaline solution hence the two DNA strands are separated
-radioactive DNA probe is added which is complimentary the VNTRs
-the DNA fragments are viewed as bands under UV light
