21 DNA technology Flashcards
what is recombinant DNA?
DNA of two different organisms that has been combined
process of making a protein using DNA technology
1) isolation: of DNA fragments that gave gene for desired protein
2) insertion: of DNA fragment into vector
3) transformation: transfer of DNA into suitable host cells
4) identification: of host cells that have taken up gene using gene markers
5) growth/cloning: of host cells
what are methods of producing DNA fragments?
conversion of mRNA to cDNA using reverse transcriptase
using restriction endonucleases to cut fragments containing desired gene from DNA
creating gene in a gene machine, based on known protein structure
how is reverse transcriptase used to isolate a gene?
cell that produces protein is selected which has lots of relevant mRNA so more easily extracted
reverse transcriptase joins DNA nucleotides with complementary bases on mRNA sequence (single stranded cDNA)
DNA polymerase makes other strand of DNA by building up complementary nucleotides on cDNA template forming required gene
how are restriction endoculeases used to produce DNA fragments?
cut DNA double strand at recognition sequence
what are two ways restriction endoculeses can cut DNA?
straight cut: between two opposite base pairs leaving blunt ends
staggered cut: leaving exposed DNA and sticky ends
how is the gene machine used to produce DNA fragments?
1) desired amino acid sequence determined, from this mRNA codons looked up and complementary DNA
2) desired sequence of nucleotide bases for gene is fed into computer and checked for biosafety
3) computer designs single strands of nucleotides (oligonucleotides) which are assembled into desired gene
4) replicated using PCR which also produces complementary strand to make double stranded gene
5) using sticky ends, gene can be inserted into bacterial plasmid which is vector
advantages of using the gene machine?
any sequence of nucleotides can be produced in a short time with great accuracy
no introns so can be transcribed and translated by prokaryotic cells
importance of sticky ends
if same restriction endonucleases used to cut DNA, fragments will be complementary and can be joined together
can be used to combine DNA of one organism with that of any other organism
what is DNA ligase used for?
once complementary stick ends have paired up, it binds phosphate-sugar framework of two sections of DNA
how is DNA fragment prepared for insertion?
addition of extra lengths of DNA
RNA polymerase binds to promoter region, nucleotide bases of promoter region attach RNA polymerase and transcription factors and begin process of transcription
terminator releases RNA polymerase and ends transcription
how is DNA fragment inserted into a vector?
same restriction endonuclease cut plasmid so sticky ends complementary to DNA fragment
joined together with DNA ligase forming recombinant DNA
how is DNA introduced into host cells?
transformation
plasmids and bacterial cells mixed in medium containing calcium ions and changes in temperature which make bacterial membrane permeable
why do not all bacterial cells possess DNA fragments with desired gene?
only few bacterial cells take up plasmids
some plasmids close up without incorporating DNA fragment
some DNA fragment ends join together to form own plasmid
what types of marker genes can be used?
antibiotic resistant marker genes - replica painting
fluorescent markers - GFP
enzyme markers - e.g. lactase, colour change
stages of PCR
1) 95c breaking hydrogen bonds so single stranded
2) 55c primers anneal to complementary bases at end of DNA fragment
3) 72c optimum temp for DNA polymerase to add complementary nucleotides along strand, beginning at primer
what is the function of a primer?
provide starting sequence for DNA polymerase to begin adding bases as it can only attach nucleotides at the end of an existing chain
prevent separate strands from rejoining
what is required for PCR
DNA polymerase (taq) - enzyme that joins nucleotides
primers - short sequence of nucleotides with bases complementary to end of DNA fragment
nucleotides
thermocycler
DNA fragment
why is taq polymerase used in PCR?
obtained from bacteria in hot springs so tolerant to heat (thermostable) and doesn’t denature at high temperatures
advantages of in vitro cloning (PCR)
rapid - small amount of dna available e.g. crime scene but also increases contaminating dna at crime scene
doesn’t require living cells - less time and effort
advantages of in vivo cloning
useful to introduce gene to another organism - once in plasmid it can go to other organisms
no risk of contamination - gene cut by same restriction endonuclease as plasmid so contaminant DNA not taken up
accurate - mutations are rare
cuts out specific genes
what are types of DNA probe?
radioactively labelled - identified using x-ray
fluorescently labelled - emit light under UV right
what are DNA probes and hybridisation used for?
to locate a particular DNA sequence (gene)
how are DNA probes used?
made with base sequences complementary to desired sequence
double-stranded DNA heated so separated
separate strands mixed with probe (cooled) which binds to complementary sequence (hybridisation)
unattached probes washed and site where probe binds identified by radioactivity of fluorescence
what is genetic screening used for?
to identify carriers of mutant allele and probability of offspring having genetic disorder
detecting oncogenes and mutations prevent tumour suppressor genes functioning, if mutated gene detected individuals at greater risk of cancer can make decisions about lifestyle and future treatment e.g. stop smoking, lose weight, mastectomy
why is personalised medicine advantageous?
allows doctors to provide care based on genotype
drugs may be more or less effective in treating a condition
can determine dose of drug needed for desired outcome
save money as not overprescribing drugs
what is genetic counselling?
research family history of inherited disease and advise on likelihood of arising in children
inform on emotional, medical, economic consequence of disease
genetic screening provides basis for counsellor’s informed discussion
why is genetic screening effective?
95% of human DNA is introns which is made up of variable number tandem repeats
probability of two individuals have same VNTRs is very low, but more closely related the more similar the VNTRs are
what is gel electrophoresis used for?
to separate DNA fragments according to size
process of gel electrophoresis?
DNA fragments placed on agar jelly and voltage applied across it
resistance of gel means large the fragment, more slowly it moves
over time, small fragments move further
DNA fragments may be labelled to identify final position
limitations of gel electrophoresis?
can only sequence DNA up to 500 bases long
larger genes must be cut into smaller fragments by restriction endonucleases
stages of genetic fingerprinting
extraction: of dna, if small can be increased with PCR
digestion: dna cut into fragments by restriction endonucleases
separation: gel electrophoresis, then immersed in alkali so double strands separated
hybridisation: complementary dna probes used to bind with VNTRs under specific conditions
development: transferred to nylon membrane
why is results of genetic fingerprinting transferred to nylon sheet?
agar gel will shrink and crash as it dries
how is genetic fingerprinting used in determining genetic relationships and variability?
paternity testing: half of genetic material inherited from each parent so each band should correspond with one of the parents
determining genetic variability within population: the more closely related the closer the resemblance of fingerprint, so if members have similar genetic fingerprints there is little genetic diversity
limitations of using genetic fingerprinting in forensic science
close match doesn’t mean suspect carried out the crime:
- dna may have been left on another innocent occasion
- dna may belong to close relative
- dna sample may have been contaminated after crime
probability that someone else’s dna matches suspect is calculated based on assumption dna randomly distributed in community but might be case e.g. religious/ethnic
how is genetic fingerprinting used in forensic science?
dna often left at crime scene (e.g. blood/semen)
establish if person likely to have been present at the crime
how is genetic fingerprinting used in medical diagnosis?
e.g. huntingtons
sample compared with people with various forms of disease
determine probability of developing symptoms and when
identify nature of microbial infection by comparing fingerprint of microbe in patient with known pathogens
how is genetic fingerprinting used in plant and animal breeding?
prevent inbreeding in farms and zoos
identify plants or animals with desirable allele for selective breeding, so increased probably of offspring having desired characteristics
determination of paternity in animals, establishing the pedigree
