6.3 -Manipulating Genomes Flashcards
what is DNA sequencing ?1
- a technique that allows genes to be isolated and read.
what did FRED SANGER do in order to sequence DNA ?
6 marks
1.single strand of DNA is obtained by heating DNA TO 95c
hydrogen bonds break leaving single strand of DNA
2.Add primer and cool to 50C, make sure to radioactivly label a base i.e. : Adenine
3.DNA polymerase will keep adding bases .
4.Termintor stops the coding
5.Electrophoresis
6. activated base pairs travel through the electrophoresis gel and then we can read the sequence .
Outline Pyrosequencing ?
6
- cut Double stranded DNA into fragments .
2.heat breaks the strands into single strands
3.add primer and incubate with
enzymes :DNA polymerase, ATP sulfuryase,Luciferase,
substrates :APS,luciferin and activated DNA
4.if activated base is incorporated into the strand then PPi is released - In the presence of APS the tap sulphurase can convert PPI–> ATP !
5.The ATP means that luciferase can work to convert luciferin —–> oxyluciferin
whats the purpose of PCR ? 1
amplify dna so it has enough to be analysed
why is Mg added in PCR ?1
cofactor for dna polymerase
what does DNA ligase do ?1
joins up fragments of DNA to build sugar phosphate backbone .
what does reverse transcriptase do ?1
- makes cDNA ( no introns) from mRNA strand .
what does restriction endonuclease (restriction enzyme ) do ? 1
-cleaves DNA at recognition sites
what do sticky ends do ? 1
- enable annealing between genes , plasmid to make recombinant DNA .
how can we introduce a vector into recipient cell ?3
- modified Virus
- Recombinant plasmid
- Gene Gun
what does a DNA probe do ? 3
it contains a sequence of DNA molecules which are complementary to DNA sequence
- so it binds to that spot on the DNA , if it’s dyed with fluorescent dye then it can be seen.
- this means that the position of that DNA sequence can be seen .
whats Annealing ? 1
- formation of Hydrogen bonds
whats electrophoresis ? what charge does DNA have ? which fragments move fastest ? how can u read the sequence ? 4
- seperation of DNA fragments based on length of strand .
Dna has a negative charge so it travels to the positive terminal .
-It puts DNA in size order to read the base sequence . - the shorter stands move faster
- read sequence from bottom ( near the positive terminal )
why can’t gene therapy treat recessive disorders ?
4
- A recessive allele doesn’t produce the correct protein
- the Doctors insert a functioning copy of the allele to make the protein where it’s lacking .
- A dominant allele makes a HARMFUL protein so adding a functioning protein, won’t do anything about the negative side effects .
- Dominant allele can’t be taken out of a cell
why is germ line therapy not allowed ?3
- alters genome of descendants without there consent
- genetic disease passed on through generations
- unpredictable
how can CRISPR technology be used to treat Dominant alleles ? 2
- precise tool is used to identify where the faulty allele is
- cut the DNA to snip it out
what are the principles of genetic engineering ?
4
- gene is obtained
- copy of the gene placed in vector
- the vector carries the gene to recipient cell
- recipient expresses the novel gene
how can you obtain a gene ?
4
2 methods explained
1-DNA Probe used to locate the gene and then restriction enzyme used to cut it out .
2-mRNA is obtained from the cells where the gene is being expressed and then Reverse transcriptase can be used to generate CDNA using the mRNA as the template .
-Then you can add primers and DNA polymerase can make cDNA into double stranded DNA which has a base sequence that codes for original protein .
How do you place the gene into a vector ?
3
- Plasmids can be optioned from bacteria
- add restriction enzyme which will cut the plasmids and produce sticky ends
- DNA ligase can join the obtained gene and Plasmid together making a recombinant plasmid
how do you get plasmid into the recipient cell ?
5
name three
1- heat shock treatment (cold/hot+cacl2) 2-electroporation -high voltage pulse 3-Electrofusion -electric fields 4-Transfection = DNA packed into bacteriophage 5-gene gun
whats the technique that makes enzymes more thermostable and allows them to be re-used ?1
immobilised
what are the two microbial culture methods ? 2
1-Batch
2-Continuos
how can bioinformatics and DNA sequencing be used to increase the effectiveness of a vaccination programme against ebola ?
DNA SEQUENCING:(high) mutation (rate) means many , strains / AW , of virus exist. It can predict the viral strain and so the vaccine will contain the correct antigen .
Bioinformatics : facilitates access to large amount of data facilitates access to data on DNA and proteins ( idea that format (of information) is universal )
can identify source of outbreak
can identify vulnerable populations vaccination program can target certain , area
