Gene Technology Flashcards
Genome
Complete set of genes in a cell
Recombinant DNA
A cell having two or more sources of DNA
Possible as genetic code is universal,degenerate and non-overlapping
5 steps in recombinant DNA technology
Isolation of genes, insertion, transformation, identification and growth
1- isolating a copy of specific DNA fragment
What are the different methods
Gene machine
Reverse transcription
restriction endonucleases
1-using reverse transcriptase
Isolated mRNA from donor cell
Add dna nucleotides and reverse transcriptase
Free dna nucleotides bind to single stranded mRNA via complimentary base pairing
Reverse transcriptase joins dna nucleotides together to form single standard cDNA molecule
Add dna polymerase to make cDNA double stranded
Advantages of using reverse transcriptase
mRNA is easy to obtain
Using mRNA means introns removed whereas genes contain both introns and exons
2-using restriction endonuclease
They hydrolyse dna at specific base sequence (either side of gene)
Regcognition sequence is usually palindromic -based pair is same whether read forwards or backwards
Cut made is blunt(used in pcr and gel electrophoresis)
Or sticky(used in transformation)
C-gene machine
Enter the desired sequence of nucleotides into a computer
Creates small sections of overlapping single strands of nucleotides
Joined to make dna
Pcr used to amplify and make double stranded
Advantages of the gene machine
Artificial genes are easily transcribed and translated by prokaryotes as they have no introns in their dna
What is a vector
DNA carrier
Insertion of genes into a vector
cut vector using the same restriction enzyme that was used to isolated dna fragments
-Produces complimentary sticky ends between ends of dna fragment and cut ends of the vector dna
-Target dna fragment anneals to vector dna by complimentary base pairs between their sticky ends
-DNA ligase used to join them at sugar phosphate backbone and forms phosphodiester bonds
Transformation
Heat shock/ electric current used to make temporary holes in cell curvaceous membrane
Bacterium takes up vector plasmid
Recombinant organism
Identifying transformed host cells
not all vectors take up target dna to become recombinant
Marker genes are added
Fluorescence - fluoresces when exposed to uv light
Enzyme markers - no colour change indicted required gene has been inserted
Identifying transformed bacteria using antibiotic resistance genes
Cells that didn’t take up plasmid are killed by both types of antibiotics
Cells that took up original plasmid are resistant to both types of antibiotics
Cells that took up the transformed plasmid are resistant to one antibiotic
Cloning in vitro via pcr
Pcr used to amplify
Several stages that automatically repeat
1 seperation - strands separate at 95 c
2 annealing -addition of primers ,primers bond 50c
3 synthesis - makes 2 dna fragments 1old 1 new
Primer
Short pieces of single stranded dna with complimentary base sequences to dna fragments
Benefits of gene tech
Develop medical applications
Better understanding of biological processes
Concerns with gene tech
Introducing herbicide resistant genes to crops
could result in transfer to wild plants
Which could become resistant to herbicides
Somatic and germ line gene therapy
Somatic - dna transfer to our normal body tissue
Germ line-dna transfer to cells that produce eggs or sperm
Limitations of somatic and germ line gene therapy
Not all cells take up new dna
Multiple treatments may be needed
Objections of gene therapy
Tech is imperfect
Denial of human rights
Potential abuse
Genetic screening using dna probes and dna hybridisation
-determine nucleotide sequence of the mutant allele that you’re trying to find
Create comp dna probe and add a marker
-many dna probes made using pcr
-dna with suspected alle is heated to separate both strands
-seperated strands are cooled in a mixture of dna probes
If strand has mutant allele probe will bind to it as bases are complimentary
-dna is washed to remove unattached probe
-remaining hybridised dna will be fluorescently binded
-dye is detected by shining light causing fragment to fluoresce
Gene probe
Short, single stranded dna molecule with complimentary base sequence to dna fragment
Genetic fingerprinting and VNTR’s
Uses the region of dna between genes called VNTR’s
Each person has 2 copies of each VNTR on each homologous chromosome
1 from each parent
Difference in length in VNTR can be identified by gel electrophoresis
DNA fingerprinting
-extract dna from the nucleus of cells
-pcr amplifies VNTR regions
restriction enzymes are used to cut dna into fragments
-must make blunt ends
-separate dna fragments using gel electrophoresis
-put dna in well and apply electrical current
-make fragments single stranded by soaking in alkali solution
-add radioactive gene probes with comp base sequences to VNTR
-identify base sequence using x ray film
Using liposomes as vectors
Using liposomes as vectors
Lipid droplets which can cross phospholipid bilayer and releases target dna into the cell
However dna does not move into the nucleus so new daughter cells will not have the functional gene
