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Biology-Corinna > 21.Recombinant DNA technology > Flashcards

21.Recombinant DNA technology Flashcards

1
Q

What is genetic screening

A

DNA probes that have code complimentary for mutation/s being investigated are fixed to glass slide
DNA being tested added to glass slide
If DNA sequence has the sequences for mutation it will hybridise to probe
Detect fluorescence

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2
Q

Why is genetic screening valuable

A

Can screen for mutant alleles, if it is in family history
Determine probabilities or children having the mutation
Detection of oncogenes-cancer
Inform individuals to help them make decisions about future treatment and lifestyle
Personalised medicine

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3
Q

What’s personalised medicine

A

Allow doctors to provide advice and health care based on individuals genotype
Some genes may mean some medicines are more effective or less effective

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4
Q

What’s genetic counselling

A

Special form of social work
Advice and info is given to help people make personal decisions about themselves or their offspring
Research of family history and chance of inheritance in their children

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5
Q

What is recombinant DNA

A

DNA of two different organisms combined together

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6
Q

What is transgenic

A

Organism with the recombinant DNA

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7
Q

What is the basic stages of gene transfer and cloning

A

1-isolation of dna fragments with desired gene
2-insertion of DNA fragment into vector
3-transformation transfer of DNA into host
4-identification of host cells that have taken up gene using gene markers
5-growth/cloning of population of host cells

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8
Q

what are the three ways that DNA fragments can be produced

A
  • conversion of mRNA to cDNA using reverse transcriptase
  • using restriction enzymes to cut fragments containing the desired gene from DNA
  • creating gene in gene machine (usually based on known protein)
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9
Q

how is complementary DNA made using reverse transcriptase

A
  • cell that rapidly produces protein that was selected
  • cells have large quantities of relevant mRNA which is easily extracted
  • reverse transcriptase is then used to make DNA from RNA, the DNA is known as complementary DNA (cDNA) as made up of nucleotides complementary to the mrna
  • enzyme DNA polymerase is used to build up the other complementary DNA strand that is complementary to nucleotides on cDNA
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10
Q

How are endonucleases used to form fragments of DNA

A

Break the phosphodiester bonds between nucleotides sometimes the cut is straight across other times it is staggered, if staggered the recognition sequence is palindromic leaving sticky ends

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11
Q

where do endonucleases come from?

A

bacteria cells that in order to defend themselves from viral infection use endonucleases to cut up viral DNA

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12
Q

what is the gene machine

A
  • manufacturing of genes in a lab
  • desired sequence of nucleotide bases of gene is determined, amino acid sequence determined, codons and triplets are found
  • desired sequence of nucleotide bases fed into computer
  • sequence checked for biosafety
  • computer design oligonucleotides
  • olignucleotides assembled by adding one nucelotide at time in required sequence
  • oligonucleotides join together making gene
  • gene replicated using the polymerase chain reaction
  • constructed complementary strand to make double stranded gene
  • using sticky ends inserted into bacterial plasmid
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13
Q

whats the importance of sticky ends

A

provided the same restriction endonuclease is used we can combine DNA of one organism with that of another organism

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14
Q

what does in vivo mean

A

by transferring fragments into host cell using vector

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15
Q

what does in vitro mean

A

using polymerase chain reaction

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16
Q

how are sticky ends produced

A

recognition site is cut using restriction endonucleases, if recognition site is cut in staggered fashion ends of DNA will have a few unpaired nucleotides forming sticky ends

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17
Q

how is DNA fragments prepared for insertion

A

addition of extra lengths of DNA called promoter where RNA polymerase can bind to, addition of extra lengths called terminator lead to the detachment of RNA polymerase and so transcription stops

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18
Q

how is DNA fragment inserted into vector

A
  • plasmid is most commonly used vector
  • same endonuclease used to cut out fragment of DNA is used to cut the plasmid loop at one of the antibiotic resistant genes
  • sticky ends of DNA fragment complementary to sticky ends of plasmid, when mixed they may become incorporated into plasmid
  • when incorporated DNA ligase reforms the phosphodiester bonds
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19
Q

how is DNA of vector introduced into host cell

A

when bacterial cells and plasmids put into a medium containing calcium ions and by fluctuating the temperature bacterial membrane becomes more permeable allowing plasmids to pass through into the cytoplasm

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20
Q

what are some reasons that not all plasmids will contain the desired gene

A
  • only few bacterial cells take p plasmids when mixed together
  • some plasmids will have closed up without incorporating DNA fragment
  • sometimes DNA fragment ends join together to form its own plasmid
21
Q

why are marker genes identifiable

A

resistant t antibiotic
make fluorescent protein easily seen
produce enzyme whose action can be identified

22
Q

how are antibiotic resistant marker genes used

A
  • DNA from cell which codes for desired protein, it is inserted into middle of gene coding for antibiotic resistance, there gene for resistance incomplete and so will no long be resistant
  • when grown on culture of antibiotic these cultures will die
  • replica plating used to identify living cultures that contain required gene
23
Q

how are fluorescent markers used

A

transfer of gene from jellyfish into plasmid
gene produces green fluorescent protein, gene being cloned is inserted into this GFP gene, any bacterial cell containing cloned gene will not produce GFP and will not fluoresce. As not killed don’t need replica plating, obtain the cultures that don’t fluoresce

24
Q

how are enzyme markers used

A

gene that prodcues enzyme lactase, lactase turns a particular substrate blue, required gene inserted into gene that makes lactase. if plasmid with required gene is present in bacterial cell the it will not produce lactase wo when grown on colourless substrate will not turn it blue

25
Q

what is the polymerase chain reaction

A

method of copying fragments of DNA, process is automated
1-separation of DNA strand
2-annealing of primers
3-synthesis of DNA

26
Q

what happens in stage one of polymerase chain reaction

A

DNA fragments, primers, and DNA polymerase put into thermocyler, temperature is increased to 95 degrees, two strands of DNA fragemtns to separate due to breaking of hydrogen bonds between two DNA strands

27
Q

what happens in stage 2 f polymerase chain reaction

A

mixture cools to 55 degrees, primers anneal to complimentary bases of ends of DNA fragment. Primers provide starting sequence for DNA polymerase to begin DNA replication

28
Q

what happens in stage three pf polymerase chain reaction

A

temp increased to 75 degrees, which is optimum temp for DNA polymerase to add complementary nucelotides along each of separated strands, begins at primer and continues on until reach end of chain.

29
Q

advantages of in vitro

A
  • extremely rapid

- does not require living cells only base sequence

30
Q

advantage of in vivo

A
  • useful when introducing gene into another organism
  • no risk of contamination-due to endonucleases creating sticky ends complimentary to each other
  • very accurate-DNA copied has very few errors
  • cuts out specific genes
  • produces transformed bacteria that can be used to produce of large quantities of gene product
31
Q

benefits of recombinant DNA technology

A
  • micro organisms to control for pollution eg break up or digest harmful gases
  • genetically modified plants transformed to produce specific substance in organ of plant, harvested and desired substance extracted
  • genetically modified crops engineered for financial and environmental advantages
  • genetically modified animals produce expensive drugs, antibiotics and hormones cheaply
32
Q

drawback of recombinant DNA

A
  • manipulation of DNA may have consequences in metabolic pathway of cell
  • genetically modified bacteria have antibiotic resistance gene might spread to harmful bacteria
  • consequences of gene manipulation into wrong hands
  • eugenics leading to selection of one race over the other
33
Q

what is a DNA probe

A

short single strand of DNA that has some sort of label attached to be able to identify it
-radio active and fluorescent probes

34
Q

what are radioactive probes made of

A

nucleotides and isotope 32P identified using x-ray film

35
Q

what are fluorescent probes made of

A

DNA that fluoresce under certain conditions

36
Q

how are DNA probes used to identify certain alleles of genes

A
  • DNA probe that has complementary bases to the base sequence of allele of gene
  • double stranded DNA is treated to separate the two strands
  • separated DNA strands mixed with probes, which binds to the complementary bases of one strand, DNA hybridisation
  • site of where probe binds is identified by the fluoresce or radioactivity
37
Q

what is DNA hybridisation

A

when a section of DNA or RNA is combined with a single stranded section of DNA which has complementary bases

38
Q

process of hybridisation

A
  • two strands DNA separated by denaturation (heating until separate into complementary single strands
  • when cooled complementary bases on each strand anneal with each other forming double strand
39
Q

locating specific alleles of genes

A
  • determine sequence of nucleotide bases of mutant allele trying to locate
  • fragment of DNA produced has complementary base seq. to mutant allele
  • copies of DNA probe using polymerase chain reaction
  • attach marker
  • DNA from person separated using heat
  • separated strands cooled in mixture containing DNA probes
  • if DNA contains mutant allele one of probes is likely to bind to it as complementary bases
  • DNA washed to remove excess DNA probes
  • remaining hybridised probes will be fluoresce under light
40
Q

what is genetic fingerprinting

A

relies on the non-coding bases of DNA, variable number tandem repeats (VNTRs)
every individual has different lengths and those more closely related will have similar VNTRs

41
Q

what is gel electrophoresis

A
  • used to separate DNA fragments, place on agar gel and voltage passed across it
  • resistance of gel means that larger fragments move more slowly
  • over fixed time smaller fragments move further
  • if DNA fragments are labelled with radioactive DNA probes final positions can be determined by placing an x-ray film over agar gel
  • anything over 500 bases must be cut into smaller fragments by endonucleases
42
Q

what are 5 stages of making of genetic fingerprint

A

extraction of DNA, sample made larger using polymerase chain
digestion of DNA, cut into fragments using endonucleases
separation of fragments by size using gel electrophoresis, immersed in alkaline solution to separate double strand
hybridisation of probes to target DNA sequences
development under x ray film to reveal pattern of bands

43
Q

how are results of genetic fingerprinting interpreted

A
  • two samples of blood eg one from suspect and one at scene
  • visually assessed, if appear to be match pattern of bars passed through automated scanning machine which calculates length of DNA fragments from bands
  • using data obtained by measuring distances travelled during electrophoresis
  • odds calculated of someone else having identical finger print
  • closer the match of two patterns greater probability that two sets of DNA come from same person
44
Q

what are uses of genetic fingerprinting

A

genetic relationships and variability
forensic science
medical diagnosis
plant and animal breeding

45
Q

how is genetic fingerprinting used in genetic relationships and variability

A
  • used to help resolve questions of paternity
  • offspring inherit half of DNA from father and half from mother therefore each band will correspond with either mum or dad
  • determining genetic variability within population, more closely two individuals are related closer the resemblance of genetic fingerprints, population with similar genetic fingerprints has little genetic diversity
46
Q

how is genetic fingerprinting used in forensic science

A

DNA from crime scene, blood of violent crime, semen from rape, hair from robbery
genetic fingerprinting can establish whether suspect had been to crime scene
but does not determine if suspect did it as may have been at scene on another occasion or a closely related relative was there instead
probability that someone else’s DNA may match has to be carried out

47
Q

how is genetic fingerprinting used in medical diagnosis

A
  • detect and diagnose diseases such as huntingtons diseases
  • huntingtons has three base sequence AGC at end of gene chromosome 4, those with more than 38 repeats more likely to get the disease and if over 50 repeats early onset
48
Q

how is genetic fingerprinting used in plant and animal breeding

A

used to prevent undesirable inbreeding
-identify plants and animals with particular alleles, breed those with favourable characteristics, increase allele frequency-pedigree breed

Biology-Corinna (22 decks)

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