Topic 7 Flashcards
(14 cards)
genome
total of all genetic material in an organism (contains coding exons and non-coding introns)
PCR
Polymerase chain reaction
Method of amplifying a sample of fragments of DNA
DNA strand is separated
DNA fragments to be copied, primers (short nucleotide sequences), four nucleotide bases and DNA polymerase (thermostable TAQ polymerase from bacteria) are placed in a PCR machine at 95°C
Breaks H bonds between the bases for replication
Addition (annealing) of the primers
Mixture is cooled to 55°C
Enables primers to anneal to the complementary bases of single DNA strands
Synthesis of DNA
Temperature increases to 72°C
DNA polymerase joins the nucleotides together forming a complementary strand
DNA sequencing
can predict the amino acid sequence of proteins for identifying faulty genes that cause disease
Terminator bases are added which halt the addition of more bases which results in varying lengths of DNA fragments and allows prediction of base sequence
Gel electrophoresis is used to separate the DNA fragments by length using an electric current
DNA profiling/ fingerprinting
DNA fragments are cut by restriction endonucleases to form shorter fragments
Gel electrophoresis separates DNA fragments by length using an electric current
Alkaline buffer denatures DNA fragments to expose base pairs for Southern blotting
DNA probes are added and fluoresce
Same micro/ mini-satellites appear in the same positions but with different number repeats so they have a unique pattern
transcription factors
proteins which bind to DNA in the nucleus and affect the process of transcribing the genetic material into mRNA
promoter sequences
transcription factors bind to specific regions on DNA to stimulate transcription
enhancer sequences
transcription factors bind to specific regions on DNA to make it more/ less available to RNA polymerase by changing structure of chromatin to either stimulate/ prevent transcription
epigenetic modification
inheritable changes of gene function without changes of DNA base sequences due to environmental factors e.g. DNA methylation, histone modification
cell differentiation
unspecialised cells switch genes on/ off to become specialised due to epigenetic modification
iPSC
how they are made
+ (2)
- (1)
induced pluripotent stem cells
differentiated adult cells reprogrammed by the artificial addition of new genes to become pluripotent and able to differentiate into many types of cell
+ overcomes use of embryonic stem cells
+ no risk of infection if individual provides the adult cells
- hard to make cells pluripotent and harder to differentiate into desired specialised cell
recombinant DNA
new DNA produced by the artificial combination of more than one organism’s DNA by genetic engineering
forming recombinant DNA
- Isolating desired DNA gene in two ways:
• Restriction endonuclease cuts the desired DNA strand at specific sites sometimes forming sticky ends so it is easier to attach new DNA
• Reverse transcriptase catalyses mRNA to produce complementary DNA (cDNA) - Plasmid DNA from bacteria are used as a vector and are cut by the same restriction endonuclease (or a virus)
- Inserting gene into the vector using DNA ligase to produce recombinant DNA
- Vector is inserted into host bacterial cell (with existing plasmids removed) to form a modified bacterium that can produce the desired protein
• Gene guns: DNA shot at high speeds into cell
• Liposome wrapping: DNA is wrapped in liposomes so are able to pass through the cell membrane
identifying recombinant cell
Marker genes may be antibiotic resistant or produce a fluorescent protein
Replica plating: growing identical patterns of bacterial colonies on different media as some can survive the presence of specific antibiotics or only survive with a specific nutrient
- Bacteria, some containing recombinant DNA with marker gene, grows on a master plate with a complete medium
- Sterile velvet is pressed onto master plate producing an imprint
- Second (replica) plate lacks nutrients required by marker gene and picks up imprint when velvet presses on its surface
- Incubated and only colonies not genetically modified survive
- Replica plate compared to master plate so genetically modified colonies are identified as they are missing on replica plate
transgenic plants
transfer of a gene from plant to another using Ti plasmid to transfer beneficial genes e.g. pesticide and flood resistant, nutrient value
