Manipulating genomes Flashcards
(28 cards)
what must happen to DNA to produce a DNA profile
extracted (and many copies made using PCR)
digested (broken into fragments) using restriction endonucleases
separated using electrophoresis
hybridized with probes (which bind to fragments and enable them to be visualised)
visualised in banding patterns (bars)
describe the PCR DNA profiling purpose and process
purpose: DNA amplification (copying)
process: DNA sample placed in thermocycler
95°: breaks H2 bonds in DNA, splitting it into 2 strands
55°: primers bond to end of each DNA strand
72°: Taq DNA polymerase joins free nucleotides to each strand
describe the electrophoresis DNA profiling purpose and process
purpose: separation of DNA fragments
process: DNA fragments are placed at the end of a gel plate, a positive electrode is at the opposite end of the plate.
DNA moves towards the positive electrode when a current is applied (all DNA fragments have phosphate groups with negative charge)
Longer fragments move slower, shorter fragments move faster
DNA fragments therefore separated into bands based on size
suggest why electrophoresis is sometimes described as similar to chromatography (2 marks)
in both procedures molecules are separated by size,
some molecules are slowed (by stationary phase in chromatography) more than others
describe 2 similarities and 2 differences between semi-conservative replication of DNA and PCR (4 marks)
Similarities:
each new DNA molecule consists of one old (template) strand and one new strand,
free complementary nucleotides are joined to a template strand
differences:
only short fragments are replicated in PCR,
whereas entire chromosomes are replicated naturally,
PCR requires primers to be used,
the DNA helicase enzyme separates strands in nature, whereas temperature cycling controls the process in PCR
how is DNA sequenced
PCR is conducted
however, some of the free nucleotides in PCR have been modified- when they bound to DNA strand they terminate polymerisation- they are fluorescently coloured, all bases have different colours
new DNA strand stop growing whenever a terminator base is added- PCR is interrupted
this results in every possible chain length being produced
lasers detect the final base of each chain
sequence of DNA bases can therefore be worked out
what can DNA sequencing be used for
disease analysis, classification, genotype-phenotype relationships, synthetic biology
describe how DNA sequencing can be used for disease analysis
particular gene variants can be sequenced and linked to the risk of inheriting certain diseases, sequencing pathogen genomes enables identification of antibiotic resistance bacteria , pinpointing genetic markers for vaccines, and identification of targets for drugs
describe how DNA sequencing can be used for classification
identifying species by using DNA barcodes, studying evolutionary relationships by comparing similarities and differences between species vase sequence
describe how DNA sequencing can be used for genotype-phenotype relationships
amino acid sequences do not always match those predicted from base sequences- several phenotypes are possible from same genotype. Knowledge of both amino acid and base sequences enables comparisons to be made
describe how DNA sequencing can be used for synthetic biology
genetic engineering requires knowledge of base sequences
explain the use of sequencing in
A: analysis of disease risk
B: classification (4 marks)
A: particular allele base sequences are associated with certain diseases,
people can be screened for the base sequence
B: particular base sequences (barcodes) are unique to species,
similarities and differences in base sequences indicate the relatedness of different species
outline the differences and similarities between the use of PCR for amplification and PCR for sequencing (4 marks)
similarities:
both use DNA polymerase,
both use free nucleotides
Differences:
PCR for sequencing includes terminator bases,
different lengths of DNA are produced when sequencing,
amplification use thermocycler, but sequencing uses capillaries/ slides
suggest why protein amino acid sequences sometimes differ from those predicted from DNA sequences (3 marks)
mRNA can be modified after transcription,
RNA splicing/ introns removed from DNA,
proteins can be modified after translation
how is restriction endonucleases used in obtaining a desired gene
gene is cut from its source DNA
cutting the gene and plasmid with the same enzyme produce 2 sets of sticky ends with complementary base pairings, enabling the gene to be inserted into the plasmid
how is reverse transcriptase used in obtaining a desired gene
mRNA (transcribed from the desired gene) is extracted from cells. Reverse transcriptase is used to convert mRNA to cDNA (a single strand of complementary DNA)
what vectors are used to transfer the gene into the organism that is being modified
plasmids: circular bacterial DNA; (most common), bacterial artificial chromosomes (BACs) are synthetic structures based on plasmids. DNA ligase used to seal gene into plasmid
Viruses: eg bacteriophages, which naturally infect bacterial cells
what is the process if transformation, what methods are used
vector containing donated gene must be transferred into recipients cells
culture heating
electroporation
electrofusion
viral transfer
agrobacterium tumefaciens infection
give the organism used and key points for culture heating, electroporation and electrofusion
culture heating: bacteria, bacterial cell membranes become more permeable when heated in a calcium-rich solution. Plasmids are then able to diffuse into cells
Electroporation: bacteria and unicellular eukaryotes, electric current disrupts cell membrane, enabling plasmids to enter
electrofusion: plants, electric currents enable the cell and nuclear membranes of 2 different cells to fuse
give the organism used and key points for viral transfer and agrobacterium tumefaciens infection
viral transfer: plants bacteria and animals, viruses naturally infect cells and this mechanism can be exploited to insert DNA directly into target cells
agrobacterium tumefaciens infection: plants, A. tumefaciens naturally infect plant cells and can be used to introduce recombinant plasmids
explain why in genetic engineering it is important to use same restriction endonuclease to cut both the gene being transferred and the plasmid vector (2 marks)
restriction enzymes cut at specific recognition sites,
the gene and the plasmid must have complementary sticky ends to be able to join together
suggest the advantages of genetically modifying a crop to be herbicide resistant ( 2 marks)
the herbicide will kill only weeds that are competing with the crop for resources,
crop yield will increase and food prices will remain low
what are the types of genetic engineering and ethical issues with them
genetically modified microorganisms: use of technology for biological warfare
pest resistance in plants: GM plants could produce toxins that might harm insects other than targeted pests
pharming ( producing human medicines from GM animals): is animal welfare compromised? will it damage health of animals
patenting (legal ownership of GM technology): how available will technology be for those it might benefit? companies are able to patent techniques and GM organisms
which cells are targeted, give examples and limitation for somatic cell therapy
cells targeted: human body cells
Examples: haemophilia, cystic fibrosis, immune diseases
Limitations: possible risk of additional health problems, introduced genes are non-heritable, requires repeat treatments
