Gene technologies Flashcards
What is the purpose of PCR
(Polymerase chain reaction)
to amplify quantity of DNA
what are the 4 items required for PCR
-DNA sample to be amplified
-excess of 4 nucleotide bases (to synthesise new DNA strand)
-Tag DNA polymerase- bind nucleotides together
-Primers- provide starting sequence for Tag
Stage 1 PCR
Separating DNA Strands
Temp: 90, 15s
Unzips polynucleotides by breaking H bonds between NCB
What is the name and function of computer used in PCR
Thermal cycler
-Varies temp at precise time intervals
Stage 2 PCR
Anneling Primers
Cooled to 55 degrees, 25s
Primers bond to 3’ end of DNA strands using complementary base pairing
Stage 3 PCR
Synthesis
Heated to 75 (optimum temp for Taq), 80s
-Adds nitrogen containing bases to primers, building up complementary DNA strands
-Phosphodiester bonds form between adjacent nucleotides
-> Double stranded DNA identical to the original sequence
Uses of PCR
-Forensic science
-DNA profiling in paternity testing
-DNA amplification of DNA extracted from fossils
Similarities and differences between PCR and SCR
+free nucleotides
+ one new, one old strand in each molecule of DNA
-PCR produces only short strands (whole chro in SCR)
-PCR requires primers
-PCR requires high temps to separate DNA strands (DNA helicase in SCR)
Intron
non-coding DNA
exon
coding DNA- for ppc
What are variable number tandem repeats
Patterns of repeated adjacent nucleotides
why does Tag DNA polymerase not denature at 90 degrees
it has covalent bonds and disulphide bridges
Use of VNTRs
- genetic fingerprinting
-inheritance matching (more distant, less relatedness)
-Essential for forensic science
what is the function of electrophoresis
preparation and analysis of DNA for sequencing
How to DNA fragments separate during electrophoresis
-DNA fragments put into wells in aragose gel
-Buffer solution added
-Current applied
-Since DNA fragments are - (phosphate groups), they move to positive end
-Shorter fragments of DNA move faster and further
How can DNA bands be visualised following electrophoresis
Using fluorescent dye and a UV light
Examples of GE
Production of human insulin
Human growth hormones to treat dwarfism
What is the def of GE
the manipulation of an organism’s DNA
1 stage of GE: Gene Isolation
-Gene is isolated using restriction enzymes
-mRNA extracted from cells
-Reverse transcriptase produces single stranded complementary DNA (cDNA)
-DNA polymerase then produces strand complementary to cDNA
-end up with double stranded DNA
+double stranded DNA has no introns as produced from mature mRNA
2 stage of GE: Insertion of gene into vector
-Vector eg. plasmid cut with same restriction enzymes
-Both plasmid and recombinant gene has complementary sticky ends
- They are annealed
-DNA ligase forms phosphodiester bonds between nucleotides of plasmid and recombinant gene-> recombinant plasmid
3 stage of GE: Uptake of recombinant gene
-Put in Ca2+ solution and heat
-CSM becomes more permeable so recombinant plasmid taken up by bacteria
-Forms transgenic bacteria
Identifying transgenic bacteria
-Reporter gene is also inserted into bacterium
-Recombinant gene is usually inserted into reporter gene so that it does not work
-cells not displaying reporter gene property have taken up gene
Restriction enzymes
-Cuts DNA strand at specific site
-site complementary to active site of enzyme
-produce sticky ends
What is meant by palindromic
sequence of bases on one strand is reversed on the other strand
GE in cows
Lysozyme- antibacterial properties
GE in goats
antithrombin- prevents blood clotting
Advantages of using transgenic plants to produce human proteins
-low production costs
-unskilled labour sufficient to maintain crops
-minimal risks of contamination of human pathogens
Crops have been genetically modified to:
-resistance to herbicides
-produce insecticides
-produce vitamin A
How genes are inserted into GMC
- T-DNA, Ti plasmid inserted into bacterium
-Gene gun- DNA coated gold particles integrates into plant chro.
What is a knockout mouse
lab mouse where researchers have inactivated an existing gene by replacing it or disrupting it with an artificial piece of DNA
why are knockout mice useful
provide valuable clues about what that gene normally does.
have been used to study cancer, obesity, diabetes, arthritis etc
Disadvantages of knockout mice
-15% of altered embryos cannot grow into adult mice
-knocking out a gene may fail to produce an observable change
RNA splicing
-pre-mrna formed at the end of transcription
-contains both introns and exons
-introns are removed
-exons are spliced together
-mature mRNA is then translated to produce ppc
Alternative splicing
-some introns retained within mature mRNA and exons can be joined in different combinations
-1 gene can code for many proteins
advantages of alternative splicing
increases genetic diversity
purpose of RNA interference (iRNA)
control gene expression ie. can silence genes
Small interference RNA (siRNA) vs microinteference RNA (miRNA)
siRNA:
must be fully complementary to mrna
more precise
double stranded
miRNA:
binds in many places in mRNA so only requires partial complimentary pairing
less precise
single stranded
How iRNA works
-double-stranded iRNA binds to dicer(endonuclease)
-dicer cuts iRNA into short sections
-short iRNA binds to argonaute protein
-1 strand of iRNA (guide strand) remains bound to argonaute protein to form RISC(RNA-induced-silencing-complex)
-RISC complex binds* to mature mRNA
-prevents translation of mRNA
*cleavage catalysed by argonaute protein
Gene therapy process
-adds functional therapeutic genes to cells with defective genes within them
-allows genetic disorders to be treated
2 main problems with gene therapy
-getting functional genes into cell
-making functional genes work once they’re in the cell
Severe combined immunodeficiency disease (SCID) causes
Faulty ADA allele
-ADA essential for proper functioning of immune system so no cell mediated response etc
Delivery methods for functional therapeutic genes (FTG)
-Viruses eg. adenovirus
-Liposomes i.e. FTG wrapped in lipid molecules. Then, sprayed into nostrils, cross csm and enter cells of the lung
both ex vivo delivery methods
Difficulties with viral vectors
-difficult to control where the virus inserts the gene
-if gene inserted into gene that controls cell division, it can cause cancers
Germline gene therapy
adds to FTG into fertilised ovum
ensures all cells of organism will contain the FTG
so FTG can be passed on to next generation
Why are stem cells ideal for gene therapy
-unspecialised
-divide by mitosis to form clones
-pluripotent, so stem cells will develop into any tissue required
Ethical complications of germline
-Playing as god
-negative effects on future generations
-ethical debate- changing genomes of unborn children
-abuse of principle- designer babies?
