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BIO > Recombinant DNA > Flashcards

Recombinant DNA Flashcards

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1
Q

Uses of recombinant DNA technology

A

Combining different organism’s DNA allows scientists to manipulate + alter DNA to improve industrial process + medical treatment

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2
Q

Why does recombinant DNA work?

A

The genetic code is universal = transcription/translation occur by same mechanism and = the same aminos across all organisms

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3
Q

3 methods of DNA fragmentation

A

1) Reverse transcription
2) Restriction endonuclease
3) Gene machine

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4
Q

Describe DNA fragmentation with reverse transcriptase

A
  • Found in HIV and it makes DNA copies from mRNA
  • Cell that naturally produces protein of interest is selected
  • Cells should have large amount of mRNA for the protein
    -mRNA acts as a template and free nucleotides with complementary bases align and are joined by the reverse transcriptase
  • Single-stranded cDNA is made
  • DNA polymerase used = double stranded
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5
Q

Advantage of reverse transcriptase

A
  • cDNA is intron free as it is based of mRNA template
  • mRNA of interest available to make cDNA
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6
Q

Disadvantage of reverse transcriptase

A

Most steps = more time consuming + difficult

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7
Q

Describe DNA fragmentation with restriction endonuclease

A
  • Occur naturally in bacteria as a defense mechanism = enzyme that cut up DNA
  • Enzyme has complementary AS to DNA bases =recognition sequences = each enzyme cuts at a specific location
  • Some enzymes = cut in same location in double strand = blunt end
  • Other enzyme = cut to create staggered ends = exposed DNA bases = palindromic = sticky ends
  • Sticky ends join to DNA with complementary base pairing
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8
Q

Advantages of restriction endonuclease

A

Sticky ends make it easier to insert DNA

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9
Q

Disadvantages of restriction endonuclease

A

Still contains introns

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10
Q

Describe DNA fragmentation with gene machine

A
  • Created in lab using computerized machine
  • Scientist examine protein of interest to identify amino acid sequence = work out mRNA/DNA sequence from it
  • Enter DNA sequence into computer to check for biosafety of protein
  • Computer creates small sections of overlapping single strands of nucleotides = oligonucleotides
  • Oligonucleotides joined = DNA
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11
Q

Advantages of gene machine

A
  • Quick
  • Accurate
  • Intron free
  • Can design exact DNA fragments wanted with sticky ends + labels + preferred codons
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12
Q

Disadvantages of gene machine

A

Need to know the amino acid sequence

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13
Q

Types of fragment cloning

A

1) In vivo cloning
2) In vitro cloning

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14
Q

Steps of in vivo cloning

A

1) Inserting DNA fragment into vector
2) Transform host cells with the vector
3) Identifying transformed cells
4) Growing clones of host

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15
Q

Describe modification of DNA fragment before insertion

A
  • DNA fragment must be modified to ensure transcription
  • Promoter region: Sequence of DNA added to start of fragment = binding site for RNA polymerase = transcription
  • Termination region: Sequence of DNA added to the end of fragment = RNA polymerase detach = stop transcription = only 1 gene copied into mRNA at a time
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16
Q

Describe insertion of DNA into vector in in vivo cloning

A
  • Same restriction endonuclease used to cut plasmid
  • Same sticky ends
  • DNA fragment sticky ends are complementary to plasmid sticky ends
  • Annealing = both combined using ligase enzyme = catalyzes condensation reactions = phosphodiester bonds between nucleotides
17
Q

Describe transformation of host cell in in vivo cloning

A
  • Vector to be inserted into host cell
  • Cell membrane made more permeable by added Ca2+ ions + heat shock = increased permeability
  • Vector enters host cells cytoplasm
18
Q

Reasons host cell doesn’t take up recombinant plasmid

A

1) Recombinant plasmid doesn’t enter host cell even with increased permeability
2) Plasmid rejoins before DNA fragment entered
3) DNA fragment sticks to itself rather than plasmid

19
Q

Use of marker genes

A
  • Used to identify which bacteria successfully take up recombinant plasmid
  • 3 types:
    1) Antibiotic-resistant genes
    2) Genes coding for fluorescent proteins
    3) Genes coding for enzymes
20
Q

Describe identification with antibiotic-resistant genes

A
  • Plasmid contains tetracycline + ampicillin-resistant genes
  • DNA fragment inserted = tetracycline-resistant gene disrupted = gene no longer able to create functional protein
  • Grow bacterial colonies on agar
  • Transfer to plate with ampicillin antibiotics = see which colonies are still left = ampicillin-resistant
  • Transfer to plate with tetracycline antibiotics = see which colonies left = tetracycline-resistant = not host containing recombinant DNA
21
Q

Describe identification with fluorescent markers

A
  • Jellyfish contain green fluorescent protein = GFP
  • Inserted into plasmid
  • DNA fragment inserted into middle of gene = disrupts = prevents production of GFP
  • Check under UV light = non-glowing colonies = contain recombinant DNA
22
Q

Describe identification with enzyme markers

A
  • Lactase turns substances from colorless to blue
  • Enzyme gene inserted into plasmid
  • DNA fragment inserted into middle = disrupted = prevents production of enzyme
  • Colonies grown on agar with a colorless substance = colonies that can’t turn blue = contain recombinant DNA
23
Q

Steps of in vitro cloning

A

1) PCR

24
Q

Equipment needed for PCR

A

1) Thermocycler: Allows temperature changes over and over again
2) DNA fragment that needs to be amplified
3) DNA polymerase/taq polymerase: From bacteria in hot springs = high optimum temperature
4) Primers: Complementary short sequence of single strands attached to start + end of fragment
5) DNA nucleotides

25
Q

Describe the method for PCR

A
  • DENATURING: Temperature increased to 95°C = break H-bonds = split DNA into single strands
  • ANNEALING: Temperature dropped to 55°C = allow primers to collide + attach with complementary sequence
  • SYNTHESIS: Optimum temperature is 72°C for taq/DNA polymerase which attaches complementary free nucleotides = makes new strand aligned next to each template
26
Q

Advantages of PCR

A

1) Automated = more efficient
2) Rapid = 100 billion copies of DNA made in hours
3) Doesn’t require living cells = quicker + less complex techniques needed

27
Q

What is VNTR?

A
  • 95% of DNA is introns which consist of variable number tandem repeats
  • The more closely related you are the more similar your VNTR
28
Q

What is genetic fingerprinting?

A

Analysis of VNTR DNA fragments to determine genetic relationships + genetic variability in a population

29
Q

Steps of gel electrophoresis

A

1) Extraction
2) Digestion
3) Separation
4) Hybridization
5) Development
6) Analysis

30
Q

Describe extraction

A
  • Only need a tiny amount e.g drop of blood or hair follicle
  • Extract DNA by separating from rest of cell
  • Quantity can be increased through PCR
31
Q

Describe digestion

A
  • Restriction endonuclease is used to cut DNA into smaller fragments
  • Use enzyme that is complementary to DNA before + after target VNTR
32
Q

Describe separation

A
  • DNA samples loaded into wells in agar gel
  • Agar gel placed in buffer solution with electrical voltage applied
  • DNA phosphate backbone is negative = move through gel towards positive end
  • Agar gel = resistance = smaller DNA move through gel faster = different lengths of DNA separated
  • Alkaline added to separate double strand
33
Q

Describe hybridization

A
  • Radioactive/fluorescent probes complementary to base sequence of VNTR added
  • Different probes added to singe stranded VNTR = binding
34
Q

Describe development

A
  • Transfer agar to nylon sheet to prevent drying + cracking
  • Expose nylon sheet to X-rays/UV light to visualize positions of probes = where VNTR are
35
Q

Describe analysis

A

DNA bands compared to identify genetic relationship/disease-causing gene/DNA from crime scene

BIO (20 decks)

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