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A Level Biology Paper 2 > Gene Technologies > Flashcards

Gene Technologies Flashcards

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1
Q

What probe do you use for a recessive disease

A

Probe complementary to the recessive faulty allele

Need 2

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2
Q

What probe do you use for a dominant disease

A

Probe complementary to the dominant faulty allele

Only need 1

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3
Q

Why can protein structure be used to determine evolutionary relationship

A

Closely related means similar base sequence so similar primary structure of amino acids and therefore protein structure

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4
Q

Why is it better to compare DNA base sequence than protein structure

A

Degeneracy of triplet code

Longer so more specific
DNA base sequence contains introns but protein structure doesn’t

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5
Q

Why is it better for restriction endonuclease to be complementary to TATCTGTCTAT than just TAT

A

TAT repeated too often
Longer one less likely to be complementary to wrong parts
Enzyme is complementary to a lot of DNA for TAT so produces more small fragments

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6
Q

Why can plants synthesise insect protein

A 
Genetic code is universal
The same bases exist in all organisms
Same triplet codes for same amino acid
Desired gene can be transcribed into mRNA
And translates into insect protein
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7
Q

What does recombinant DNA technology allow

A

Genes to be manipulated, altered and transcribed from organism to organism
Better understanding of how organisms work
Design new industrial processes and develop medical applications

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8
Q

3 important features of the genetic code

A

Universal: Same triplet codes for same amino acid
Degenerative: More than one base/triplet for each amino acid
Non-Overlapping: Each base only part of one triplet/read once by ribosome

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9
Q

5 steps for producing and conducting gene transfer and cloning

A 
Isolation
Insertion
Transformation
Identification
Cloning
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10
Q

Benefit of DNA being universal

A

All organisms have the same triplets coding for same amino acid
So transferred DNA can be transcribed and translates
During protein synthesis

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11
Q

3 methods of isolation

A

Conversion of mRNA to cDNA using reverse transcriptase
Restriction endonucleased to cut a fragment with desired gene
Create gene in gene machine

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12
Q

How is reverse transcriptase used in isolation

A

mRNA mixed with free DNA nucleotides
Enzyme reverse transcriptase added and mixed in
Free DNA nucleotides bind to single stranded mRNA template via complementary base pairing via reverse transcriptase
Into cDNA
Degrade mRNA with RNAase/strong alkali to leave cDNA (single stranded)
Added DNA nucleotides and DNA polymerase to make cDNA double stranded

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13
Q

Advantage of using reverse transcriptase in isolation

A

mRNA in cytoplasm already been spliced so all introns removed
mRNA easier to obtain
Use mRNA base sequence to work out sequence of exon bases without the need for splicing
If being actively expressed, cytoplasm will contain lots of mRNA (e.g insulin mRNA abundant in beta cells of pancreas)
Cells only contain 2 alleles for a gene
Bacteria don’t have enzymes to deal with introns

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14
Q

Use of restriction enzymes in isolation

A

Cut/hydrolyse phosphodiester bonds in DNA
At specific recognition sequences complementary to their active site
These are palindromic sequences (base pair reads same both ways)
If target gene has recognition sequence before and after
DNA incubated with specific restriction endonuclease
Same enzyme must be used for DNA of interest and plasmid so sticky ends match and can form complementary base pairs via H bonds

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15
Q

Where do restriction endonucleases cut

A

Specific recognition sequence complementary to active site
These are palindromic sequences
Read the same in opposite way

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16
Q

What two things do restriction endonucleases produce

A

Sticky ends

Blunt ends

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17
Q

What are sticky ends

A

Transformation

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18
Q

Is Sam amazing

A

Yes

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19
Q

What are sticky ends

A

Overhand and complementary
Same restriction endonucleases
Needed in transformation and modification

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20
Q

What are blunt end uses

A

PCR
Gel electrophoresis
Restriction mapping

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21
Q

Regulatory genes

A

Gene that regulates the expression of one or more structural genes
By controlling the production if a protein
Which regulate their rate of transcription

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22
Q

Proteome

A

Entire set of proteins that is or can be expressed by a genome, cell, tissue or organism at a certain time

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23
Q

Gel electrophoresis

A

Method for separation and analysis or DNA, RNA and protein and their fragments based on their size and charge

24
Q

Oligonucleotide

A

Small overlapping single strands of nucleotides that are assembled into the desired gene

25
Q

Advantage of gene machine

A

Easily transcribed and translated by prokaryotes as they have no introns in their DNA

26
Q

Explain how the gene machine works

A

Desired nucleotide sequence inputted into computer
Synthesis of oligonucleotides
Which are overlapped
And joined together and made double stranded by PCR
Gene is inserted into bacterial plasmid and cloned
Sequenced, those with errors rejected

27
Q

Vector

A

DNA carrier (bacterial plasmid/virus) used to transfer foreign DNA into cells

28
Q

How is gene inserted into vector

A

Use same restriction endonucleoase to isolate DNA fragment and cut open DNA vector
So that it has a promoter and a terminator
So RNA polymerase can bind before the gene of interest and release after it
Producing complementary sticky ends
Target DNA fragment anneals to vector DNA by complementary base pairing between the sticky ends
DNA ligase uses to join DNA fragment and vector DNA at the sugar phosphate backbone (ligation)
Phosphodiester bonds formed
New recombinant DNA

29
Q

Two vectors

A

Plasmid

Bacteriophage/virus

30
Q

Two ways to encourage DNA vector to be transferred into host

A

Giving bacteria a short electrical shock
Opens temporary small pores in the cell membrane

Calcium phosphate makes temporary pores in cell membrane

31
Q

Method for in vivo cloning

A

Cut desired gene from DNA desired organism
Using restriction endonuclease

Use mRNA from cell of organism
Reverse transcriptase from desired DNA

Make artifical DNA with correct base sequences
Using DNA polymerase
Cut plasmid open
With same restriction endonuclease
Producing stick ends
DNA ligase used to join 
Return plasmid to bacterial cell
32
Q

Why must organisms be identified

A

Not all vectors take up target DNA and become recombinant
Not all host cells become transformed by taking up recombinant vectors
Only transformed host will synthesis desired protein
Only want to clone transformed cells

33
Q

Marker gene

A

Allows easy identification of cells that have taken up a genetically transformed plasmid
Those that have not will not be fluorescent/identifiable

34
Q

3 ways to use marker genes

A

Antibiotic resistance
Fluorescence
Enzyme markers

35
Q

How do you identify colonies that have taken up recombinant DNA with replica painting/stamp

A

Incubate on a growth medium
Sterile velvet surface used as a stamp
To transfer cells from each colony onto replica plates
One has the normal medium but one has the condition which only those transformed can survive in
Clone those successful

36
Q

Explain the outcomes for replica painting/stamp

A

Some cells haven’t taken up plasmid
So killed by both antibiotics

Some cells have taken up original plasmid
So resistant to both types
Some taken up transformed plasmid

So resistant to one but not the other since the gene is cut and disrupted by inserting foreign DNA

37
Q

Primer

A

Short pieces of single stranded DNA
Complementary base sequence to bases at the start of DNA fragment you want to isolate
Prevent strands sticking back together
Allow DNA polymerase to attach and join free nucleotides together

38
Q

Purpose of PCR

A

Used to amplify a single DNA fragment into millions of copies
Stages are automatically repeated many times
Number of DNA molecules doubles every cycle
So rapid and efficient

39
Q

PCR

A

Polymerase Chain Reaction

40
Q

3 steps for PCR

A

Separation
Annealing
Synthesis

41
Q

Why is the PCR cooled

A

Primer won’t attach at 95 so H bonds can form

42
Q

Steps for PCR

A

Heat DNA to 95 degrees which breaks weak hydrogen bonds and separates strands
Add primers and nucleotides
Cool to 50 degrees to allow binding of nucleotides and primers
Add thermostable DNA polymerase (Taq)
Heat to 75 degrees
DNA polymerase joins nucleotides together by phosphodiester bonds
Repeat cycle many times
Amplify DNA

43
Q

Number of DNA molecules after each PCR

A

2^n

n=number of cycles

44
Q

Number of cycles in PCR

A

Log2(n)

n=number of DNA molecules

45
Q

Advantage of using a thermostable enzyme in PCR

A

Enzyme is stable even at high temperatures
So not denatured at high temperatures
Making it efficient at amplifying DNA

46
Q

Disadvantages of in vivo cloning

A

Slower - One DNA replication per cell division so limited by growth of cell

Cant be used to copy partly broken down DNA

Not very sensitive so need a large DNA sample to start with

Need to isolate DNA fragment from DNA before it can be inserted into host

47
Q

Advantages of in vivo cloning

A

Almost no risk of contamination
Cells have mechanisms for correcting errors made when copying genes so much more accurate

Can be used to clone large DNA fragments

Can be used to produce protein or mRNA from the inserted DNA as well as target DNA

48
Q

Disadvantages of in vitro cloning

A

Can only be used to copy DNA

Lacks error-correcting mechanisms so error rate higher

Cloning becomes unreliable when used to copy DNA fragments longer than 1000 base pairs

49
Q

Advantages of in vitro cloning

A

Rapid - can produce millions of copies of DNA in hours

Only replicates target DNA so no need to isolate DNA fragment from DNA

Can be used to copy partly broken down DNA

Very sensitive, only requires a small sample of DNA to start with

50
Q

Three uses/benefits of recombinant DNA tech

A

Agriculture; GE for high yield, more nutrients, pest resistance, drought and temperature resistant

Industry; GE organisms to make large quantities of enzymes quickly and cheaply for industrial processes

Medicine; GE organisms can be produced to make large quantities of drugs and vaccines quickly and cheaply

51
Q

Issues of recombinant gene technologies

A

Antibiotic resistance marker genes within GE could lead to transfer of these genes to pathogens and decrease antibiotic effectiveness
Introducing herbicide resistance genes could result in transfer to wild species when they interbreed and produce weeds resistant to herbicides

52
Q

Why are viruses or liposomes used as vectors

A

Plasmids cannot carry DNA into human cells

53
Q

Liposome

A

Lipid droplet like a vesicle that carries DNA into cell
Fuses with cell surface membrane and releases DNA into cell
DNA doesn’t move into nucleus so daughter cell won’t have functional gene

54
Q

Somatic vs germline gene therapy

A

Somatic is DNA transfer to normal body tissue
Germline is DNA transfer to cells that produce eggs or sperm

Somatic gene therapy restricted to patient and isn’t inheritable

55
Q

VNTR’s

A

Variable Number Tandem Repeats

Not the same as introns since not in the gene

56
Q

Describe how genetic fingerprinting is carries out

A

DNA extracted from sample
Cut into segments using restriction endonucleases
Leaving VNTR’s intact
DNA fragments separated using gel electrophoresis
Mixture put into wells on gel and electric current passed through
Immerse gel in alkaline solution/two strands separated
Southern blotting
DNA fixed to nylon/membrane using UV light
Radioactive probe added which is picked up by required fragments
Identified using X-ray film/autoradiography

A Level Biology Paper 2 (16 decks)

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