Recombinant DNA technology Flashcards
What is the genome
full set of genes in each cell
why are sequencing projects important
allows genome comparison between species to determine evolutionary relationships
can identify potential antigens to use in vaccines
comparisons between individuals allows development of personalised medication
What is the proteome
full range of proteins that can be coded by the genome
How can you determine proteome of simple organisms
as there are no introns in the DNA, the genome can be translated into the proteome, as all genes are coding.
What did the human genome project do
determined the sequence of bases in a human genome
How can we use the human genome project
screening for mutated sequences allows identification of genetic disorders before symptoms occur
How long did the HGP take and how long could it take now
15 years
as little as 26 hours
What is a transgenic organism
an organism that has received a DNA transfer
how can transferred DNA be translated in transgenic organisms
as genetic code and transcription and translation machinery are universal for all species
3 ways of forming and isolating DNA fragments
Reverse transcriptase
restriction endonuclease
gene machine
How can you use reverse transcriptase to isolate DNA fragments
Cell that produces target protein is chosen as has lots of mRNA for the protein
Reverse transcriptase’s attach free nucleotides together that are complementary to the mRNA strand
a single strand of cDNA is formed
DNA polymerase makes cDNA double stranded with nucleotides.
(cDNA has no introns)
What does reverse transcriptase do and where does it naturally occur
makes DNA copies from mRNA
occurs in retroviruses e.g. HIV
What do restriction endonucleases do
They cut DNA at restriction sites to give DNA fragments
they are complementary to the base sequence at specific restriction sites
some cut through at the same location in both strands of DNA to produce a blunt end
some create staggered ends with exposed bases, these are palindromic and called sticky ends as can join comp base pairs
How does a gene machine create DNA fragments
ADBOJ
amino acid sequence of specific protein is identified, then the mRNA and DNA sequence from that
sequence entered into computer to pass biosafety and security checks
computer creates small sections of overlapped DNA strands called oligonucleotides
these can join to form DNA sequence of entire gene
What does the process of PCR do
Increases the number of DNA fragments
Describe the process of PCR
HCPD
DNA fragment heated to 95 degrees so hydrogen bonds break to separate strands
mixture is cooled to 55 degrees and primers are added to anneal to DNA fragment
heat mixture to 72 degrees so DNA polymerase can attach nucleotides to form 2 new double stranded fragments
what do primers do in PCR
primers are short sequences of DNA that join DNA fragments so that DNA polymerase has a basis to attach nucleotides together
Describe the process of transformation(In Vivo)
DTPLT
DNA cut using restriction endonucleases to create sticky ends
promoter and terminator region are added to fragments for translation
the same restriction endonucleases are used to cut open plasmid to allow complementary regions for fragment to join
DNA ligase is the enzyme used to join fragment into the plasmid
recombinant plasmid transferred into bacteria via heat shock or Ca2+
What are the issues with (in vivo) transformation
not all plasmids take up foreign DNA
not all recombinant plasmids will be taken up by bacterial cells
How to identify if plasmids have been taken up by bacteria(Marker genes)
Bacteria is grown on agar plates containing antibiotics
if bacteria survives, plasmid has been taken up as plasmids contain antibiotic resistant genes
if bacteria dies, it does not contain the plasmid
recombinant plasmids will die as recombinant gene will interfere with antibiotic resistant gene
How to identify if plasmids are recombinant
GFP
DNA fragment inserted into marker gene that encodes fluorescence
plasmids will not fluoresce if recombinant as foreign fragment has been taken up and GFP wont be made
What is gene therapy
curing/treating disorders by inserting functional alleles to mask faulty ones
Process of gene therapy
healthy allele is isolated and inserted into faulty cells by plasmids
if mutant allele is recessive, a dominant allele must be inserted
if mutant allele is dominant, DNA must be inserted to silence the allele
Difference between somatic and germline gene therapy
Somatic is when alleles are altered in body cells
Germline is when alleles are altered in sex cells and are passed onto offspring ( illegal as offspring cannot consent to the therapy )
What are DNA probes
Short sections of DNA that are complementary to a known sequence e.g. a mutated allele
labelled with a fluorescent tag or radioactive tag
Why are DNA probes used
Genetic screening, to see whether an individual possesses a recessive mutated allele for a certain disorder
or to evaluate risk of developing disease such as cancer
How do DNA probes work
labelled DNA probe is mixed with denatured DNA samples from an individual
if individual has mutated allele the probe will bind to comp base sequence in one strand
this hybridised DNA is detected using radiation or fluorescence
What is DNA hybridisation
DNA sequences from different species have comp base pairs and will hybridise
What is genetic counselling after screening
providing of information and support about results from genetic screening
What is genetic fingerprinting
method used to produce a pattern of DNA bands from an individuals genome
What are VNTRs and how do they vary between individuals
Variable number tandem repeats
short repeating sequences of DNA in non coding regions
vary in length and number of repeats so probability of 2 people having same vntrs are low
How is DNA fingerprinting carried out
AFEPVDC
Extraction of certain DNA and amplification by PCR
Digestion using specific restriction nucleases into DNA fragments
separation of DNA fragments by gel electrophoresis
VNTRs are hybridised with DNA probes
the gel is developed, pattern of bands can be seen by placing on an x-ray as probes often contain radioactive tag
this reveals position of bands and banding patterns can be compared between samples
What is the process of gel electrophoresis
DNA fragments are placed on a gel plate
the fragments will move towards the positive electrode when switched on as DNA -vely charged due to phosphate groups
smaller fragments move further toward the electrode as are lighter
different sized fragments are separated into band (thicker = more fragments)
