Control of Gene Expression - Recombinant DNA Technology Flashcards
what is a genome?
complete map of all genetic material in an organism
what is bioinformatics?
collecting and analysing biological data using computers and algorithms
how is DNA sequencing carried out?
using whole genome shotgun sequencing
what is whole genome shotgun sequencing?
DNA cut into many small overlapping sections
computer algorithms align sequences
this assembles entire genome
why is whole genome shotgun sequencing used?
genome too large to do all at once
what does SNPs stand for?
single nucleotide polymorphisms
what are SNPs?
single base variations in the genome that are associated with disease and disorders
what is the proteome?
all proteins produced by a genome
why is it easier to determine the proteome of simple organisms eg bacteria?
most prokaryotes have 1 circular piece of DNA, not associated with histones
no non-coding sections of DNA
why is it useful to know the proteome of simple organisms?
helps identify genes that code for antigens - production of vaccines
why is it difficult to determine genomes of complex organisms?
hard to translate into proteome as many non-coding genes
what is the process of DNA technology?
isolation
insertion
transformation
identification
growth/cloning
what is isolation?
isolating the DNA containing the desired gene
what are the methods of isolation?
reverse transcriptase
restriction endonucleases
gene machine
how is reverse transcriptase used to isolate a gene?
select cell that readily produces desired gene - has lots of mRNA
extract mRNA
reverse transcriptase makes DNA from RNA
cDNA produced - complementary to the RNA
DNA polymerase produces more cDNA
how are restriction endonucleases used in isolation?
restriction endonucleases recognise and cut DNA at recognition sequence
what is a recognition sequence?
specific sequence of DNA bases where a restriction endonuclease will cut
what are the 2 types of ends produced while using restriction endonucleases?
sticky ends
smooth ends
what is the difference between sticky ends and smooth ends?
sticky ends - staggered cut and so can join to another sticky end if cut with same restriction endonuclease
smooth - cannot rejoin
how can the gene machine be used to isolate a desired gene?
enter desired sequence into computer
check sequence for biosafety + biosecurity.
computer produces oligonucleotides
oligonucleotides assembled into desired gene
DNA replicated using PCR
what is an oligonucleotide?
small, overlapping single strands of nucleotides
what are the advantages of using the gene machine?
any sequence can be produced
quick
accurate
no introns
what are the 2 ways to clone genes?
in vivo
in vitro
what is in vivo cloning?
transfering the DNA into a host using a vector
what is in vitro cloning?
using PCR
what is the function of DNA ligase?
joins sticky ends into a DNA sequence
what are organisms that contain recombinant DNA known as?
transgenic
how are DNA fragments prepared for insertion?
RNA polymersase attach the DNA at the promotor region
nucleotide bases of promotor attach RNA polymerase + transcriptional factors - begins transcription
at same time terminator region releases RNA polymerase
what is the promotor region?
the biding site for RNA polymerase
what is used to insert DNA into a host cell?
a vector
what vector is usually used during insertion?
a plasmid
why are plasmids the usually used as a vector?
contain antibiotic resistance genes
restriction endonucleases can usually break these to insert
why are the same restriction endonucleases used during both isolation and insertion?
so the sticky ends created are complementary to the DNA the gene is being inserted into
what is the process of insertion?
plasmid broken at antibiotic resistance gene by same endonucleases from isolation
fragment mixes with plasmid and becomes incorporated
joined properly by DNA ligase
what is the process of transformation?
plasmid and bacterial cells mixed together in medium containing Ca2+
Ca2+ + change in temp cause bacterial membrane to become permeable
plasmids enter bacteria
why will not all bacteria have the desired gene after transformation?
some DNA fragments join together to form own plasmid
some plasmids will have closed up again before incorporating gene
not all bacteria will take up plasmid
how are the bacteria containing the desired gene identified?
using marker genes
what 3 marker genes are used?
fluorescent markers
antibiotic resistance markers
enzyme markers
how are antibiotic resistance genes used in identification?
if DNA fragment inserted into gene, bacteria will no longer be resistant to antibiotics
bacteria will not grow on plate with the antibiotic
replica plating used so not all bacteria containing the gene are destroyed
how are fluorescent markers used in identification?
flourescent protein gene incorporated into plasmid
desired gene inserted into centre of this
if the gene has been taken up, bacteria will not glow
how are enzyme markers used in identification?
if desired gene inserted - gene that makes lactase disrupted
if gene incorporated, lactase not produced
test for lactase - will remain colourless if not present
what does PCR stand for?
polymerase chain reaction
what is PCR?
a method of copying/replicating fragments of DNA
describe the process of PCR
DNA fragments, primers and DNA polymerase placed into vessel in thermocycler
temp raised to 95°C to separate strands
cooled to 55°C to allow primers to attach
heated to 72°C so DNA polymerase can add complementary nucleotides to form 2 new strands of DNA
what are DNA primers?
short sequences of nucleotides that are complementary to one of the ends of the DNA fragments
what is a thermocycler?
computer that controls and varies temperature of a period of time
what is the role of primers?
tells DNA polymerase where to attach
stops the strands rejoining
what are the advantages of in vivo cloning?
useful for inserting genes into organisms
no risk of contamination
accurate
produces transformed bacteria that can be used to produce large quantities of gene product
what are the advantages of in vitro cloning?
rapid
does not require living cells
can be used with very small samples of DNA
what is a DNA probe?
short, single stranded length of DNA with label attached to make it identifiable
what is a DNA probe made up of?
complementary sequence to a mutant allele of the desired gene
what are the 2 most commonly used DNA probes?
radioactivally labelled
fluorescently labelled
what are radioactivelly labelled DNA probes made up of?
nucleotides with isotope 32P
how are radioactivelly labelled DNA probes identified?
using an x-ray film
how are fluorescently labelled DNA probes identified?
they emit light under certain conditions
when does DNA hybridisation take place?
When a section of DNA or RNA is combined with a single-stranded section of DNA which has complementary bases.
what is the process of DNA hybridization?
heat until strands separate
when cooled, complementary bases recombine
if other complementary sections of DNA present, just as likely to anneal with separated strand
when is genetic screening used?
when there is a family history of a genetic disease so potential parents can go through genetic counselling
what is personalised medicine?
specific advice and healthcare based on an individuals genotype
what is genetic counselling?
advice given to help people make personal decisions about themselves or their offspring.
what is genetic fingerprinting?
diagnostic tool used to identify people from their DNA
what is genetic fingerprinting based on?
the fact that most eukaryotes contain VNTRs
what are VNTRs?
non-coding and repeating sections of DNA that are unique to every individual
what are the 5 stages of genetic fingerprinting?
extraction
digestion
separation
hybridisation
development
what happens in extraction during genetic fingerprinting?
DNA extracted from a sample
if only a small sample available - amplified using PCR
what happens in digestion in genetic fingerprinting?
DNA cut into lots of small fragments using restriction endonucleases
sections cut out called restriction fragments
what happens in separation in genetic fingerprinting?
separated by size in gel electophoresis
southern blotting to transfer pattern onto a nylon membrane
what happens during gel electrophoresis?
DNA fragments injected into wells and electric current applied
DNA negative so attracted to postitive end of gel
DNA separated into bands according to size - smaller fragments move further towards positive
what happens during southern blotting?
thin nylon membrane laid over gel
sheets of absorbant paper laid over membrane
liquid drawn up by capillary action
fragments drawn up and fixed in place with UV light into the exact same positions as they were in before
what happens during hybridisation in genetic fingerprinting?
radioactive or fluorescent probes attached to core sequences
any probes not bound are washed off
can repeat with different probes
what happens during development in genetic fingerprinting?
probes produce pattern on light and dark bands which is unique to each individual except twins
pattern analysed
what are the applications of genetic fingerprinting?
forensics
medical problems
how is genetic fingerprinting used in forensics?
if DNA from suspect present - highly likely they were involved in the crime
how is genetic fingerprinting used in medical problems?
determine whether a person is parent of a child
can be used in immigration cases
how is genetic fingerprinting used in genetic variability in a population?
similar fingerprinting - little genetic diversity
different fingerprinting - lots of diversity
