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MANIPULATING GENOMES Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

what is DNA profiling?

A

is a process used to determine the relationship between organisms and identification

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2
Q

what are DNA profiling used for?

A

establish evolutionary relationships
paternity testing
identifying a corpse

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3
Q

how to carry out DNA purification(1 stage)

A

-isolate DNA
-Breaking (lysing) the cells and disrupting the nuclear membranes to release the DNA by using detergent and salt (detergent and the heat disrupt the phospholipid bilayer of the cell membranes and nuclear membranes, releasing the DNA)

-Using protease to denature and remove the histones on DNA
-centrifuge solution to separate content ( SUPERNATANT -includes DNA and PELLET- unwanted things)
-Precipitating the DNA using COLD ETHANOL

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4
Q

what after DNA purification? what is the purpose of it?

A

PCR - replicate DNA
Polymerase chain reaction

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5
Q

describe the process of PCR

A
  • create a solution containing DNA SAMPLE, PRIMERS, MONONUCLEOTIDE, DNA (TAQ) POLYMERASE, BUFFER
    1. put the solution in the PCR machine, increase the temperature to 95 C - to break H bonds between bases to separate the strands
    2. at 95 C PRIMERS cannot stick on the strand due to H bonds absence, so PCR decreases temperature
    3. temperature is decreased to 65 C - now PRIMERS can ANNEAL to exposed bases and form H bonds
    4. now that primer attached to exposed bases, a sugar phosphate backbone must be formed , which is done by DNA POLYMERASE.
    5. to allow dna polymerase to form the sugar phosphate backbone, PCR increase temperature
    to 72 C to allowing DNA polymerase to synthesise strands by adding mono nucleotides to the strand and form phosphodiester bonds
    6. cycle repeats
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6
Q

what’s an INTRON?

A

non-coding region of the DNA

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7
Q

what’s an EXON?

A

coding region of the DNA

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8
Q

what does STRs stand for? explain what they are

A

short tandem repeats - small amount of base pairs which are repeated multiple times, can be found along many places of DNA, inherited from mother and father, found in introns

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9
Q

what are two properties of TAQ polymerase?

A

-can withstand extremely high temps without denaturing
(thermostable)
-synthesises in 1 direction

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10
Q

what is the step after PCR called?and what is the purpose of the of this step?

A

sanger sequencing or fluorescent tag polymerase
gel electrophoresis - separate DNA fragments of DIFFERENT SIZES

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11
Q

how does gel electrophoresis work?

A

0.replicated DNA is digested and cut into pieces using restriction enzymes.

  1. use of a gel plate made of agarose (polypeptide) at liquid state
  2. insert 1 COMB to make wells during liquid state
  3. let it cool to make it solidify
  4. remove COMB
  5. place the gel plate into a tank containing buffer solution , which will cover the gel plate an the ends of it
  6. a loading dye is added to the solution containing digested DNA - loading dye is dense which will eventually dna make it sink down the well
  7. loading dye + digested dna solution are added to a well using a micropipette
  8. the DNA sample in the well are negatively charged due to the phosphate in the sugar phosphate backbone being negative.
    9.in order to make the DNA move down the gel plate, a positive charge (anode) is used on the opposite end of wells to attract DNA fragments.
    10.the smaller fragment of DNA have less bases (and appear lighter) therefore will be able to move down faster than the long fragments (which have more bases and appear darker)
  9. the smaller the reaction centre fragment ,the greatest the distance travelled from the well/starting point
  10. differences in dna fragment can be deduced by observing the distance travelled by the different dna samples. in a given time
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12
Q

what determines the width of the band?

A

well width

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13
Q

what is DNA sequencing?

A

finding the nucleotide sequence for a gene or whole genome

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14
Q

what method can be used to do DNA sequencing?

A

Sanger sequencing

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15
Q

describe Sanger sequencing

A
  • first make use of PCR to replicate fragments of DNA
  • have 4 test tubes containing : replicated DNA fragment, DNA nucleotides, DNA polymerase, TERMINATING NUCLEOTIDES (dideoxynucleotides) (each test tube will have 4 different terminating nucleotides either ATCG, primers
  • the terminating nucleotides have radioactive tag, so make use of x-ray to observe them
  • incubate the test tubes to allow DNA polymerase to form a double stranded DNA fragment
  • at any time, DNA polymerase can attach a terminating nucleotide, terminating the the process
  • the addition of terminating nucleotide is completely random so varies each time, thus producing different lengths of double stranded DNA fragments
  • when the incubation period has ended, the developing strands are separated from the template DNA.
  • the developing single stranded sterns are then separated using gel electrophoresis
    -each well will have one different terminating nucleotides
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16
Q

describe high-throughput sequencing / fluorescent tag sequencing

A
  • more efficient, rapid and less time consuming than the Sanger sequencing.
  • use of dideoxynucleotide that has a fluorescent dye
  • use of CAPILLARY ELECTROPHORESIS to separate the single stranded DNA according to their length
  • use of laser Beam to illuminate and detect the terminating nucleotides that have a unique fluorescent dye that can be detected
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17
Q

how is Sanger sequencing read?

A

user reads all four lanes of the gel at once, moving bottom to top, using the lane to determine the identity of the terminal ddNTP for each band.

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18
Q

how is fluorescent tag sequencing read?

A

a computer reads each band of the capillary gel, in order, using fluorescence to call the identity of each terminal ddNTP.
In short, a laser excites the fluorescent tags in each band, and a computer detects the resulting light emitted. Because each of the four ddNTPs is tagged with a different fluorescent label, the light emitted can be directly tied to the identity of the terminal ddNTP. The output is called a chromatogram, which shows the fluorescent peak of each nucleotide along the length of the template DNA.

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19
Q

summarise the differences between Sanger sequencing and fluorescent tag sequencing

A

dna purification
PCR to amplify DNA
SANGER SEQUENCING or FLUORESCENT TAG SEQUENCING

SS : 4 tubes, use of primers, dna polymerase, mono nucleotide and terminating mononucelotides
FTS : use of 1 tube containing all the different dideoxynucleotides, dna polymerase, primers, mono nucleotides

SS : gel electrophorus read by the user
FTS : CAPILLARY ELECTROPHORESIS read by the computed to make the sequence

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20
Q

3 ways to visualise STRs

A
  • name STRs
    -gel electrophoresis
  • electropherogram
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21
Q

what can DNA sequencing be used for?

A

-comparison between species
-variation between individuals
-evolutionary relationship

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22
Q

explain comparison between species

A

genome comparison between species to determine difference, as evolution progresses, some genes are co-opted to perform new tasks, many of the differences between organisms are not because the organisms have totally different genes, but because some of their shared genes have been altered and now work slight differently

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23
Q

explain variation between individuals.

A

only 0.1% of our DNA is not shared with others

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24
Q

what are the places on the DNA where the substitution occur knows as? what can these cause?

A

single nucleotide polymorphism
SNPs
some have no effect on protein, some can alter a protein
some can alter the way a piece of RNA regulates the expression of another gene

25
Q

what is DNA methylation?

A

the process of adding methyl groups at the the CpG sites - which are locations of the DNA where CYTOSINE is next GUANINE in the DNA chain
methyl groups are added to CYTOSINE by the ENZYME SNA METHYLTRANSFERASE

26
Q

what does DNA methylation cause?

A

IT INHIBITS TRANSCRIPTION

27
Q

how does DNA methylation inhibit transcription?

A

1)by stopping transcription factors from binding to the DNA section and therefore preventing RNA polymerase binding

2)methyl groups attract proteins that condense the chromatin, making the gene inaccessible for transcription

28
Q

what is meant by demethylation?

A

the removal of methyl groups from DNA

29
Q

what does the methylation enable?

A

transcription of gene

30
Q

what is genetic engineering?

A

its the process of combining DNA from different organism into another, transferring one organism gene into another organism

31
Q

what is the purpose of genetic engineering?

A

to modify the genes to enhance the capabilities of the organism beyond what is normal.

32
Q

how is one gene transferred form one organism to another?

A

using a vector such as plasmid or atteaned/ weakened vector

33
Q

what are the steps of transferring one gene to another organism?

A

the required gene is obtained
a copy of the gene is placed inside a vector
the vector carries the gene into a recipient cell
the recipient expresses the novel gene

34
Q

how is the required gene obtained?

A

-mRNA is obtained from cells where the gene is expressed
-reverse transcriptase catalyses the formation of a single strand of cDNA using the mRNA as template
- addition of primers + dna polymerase can convert cDNA into a double stranded length of DNA

35
Q

what can scientist do the gene if they know the nucleotide sequence?

A

can synthesise the gene by using an AUTOMATED POLYNUCLEOTIDE SYNTHESISER

36
Q

what can scientist do to the gene if they know the sequence of the gene?

A

amplify the gene by using PCR

37
Q

what is used to locate a gene in the genome?

A

DNA probe

38
Q

restriction enzyme function

A

cut the gene required

39
Q

placing the gene into a vector method:

A

-plasmid from bacteria is obtained and is cut using a restriction enzyme AT SPECIFIC RECOGNITION SITES
- the cur plasmid has an exposed unpaired nucleotide bases classed STICKY ENDS
- if free, complementary base pairs to the sticky end are addend to then ends of the gene to be inserted, GENE AND PLASMID SHOULD ANNEAL
- annealing is catalysed by DNA LIGASE

40
Q

Getting the vector into the recipient cell

A

in order to make DNA pass through the cell plasma membrane VARIOUS METHOD ARE USED:
- heat shock treatment
-electroporation
-electrofusion
-transfection

41
Q

describe heat shock treatment

A

bacterias that are subjected to alternative periods of cold and heat in the presence of calcium chloride, their wall become more porous and allow in the recombinant vector
This is because the positive calcium ions surround the negatively charged parts of both dna and phospholipids, THUS REDUCING THE REPULSION BETWEEN THE FOREIGN DNA AND HOST CELL MEMBRANE

42
Q

describe electroporation

A

high voltage pulse supplied to the cell to disrupt the membrane

43
Q

describe electro fusion

A

electrical fields help to introduce DNA into cells

44
Q

describe transfection

A

dna can be packaged into a bacteriophage, which can then be transfect the host cell

45
Q

where are reverse transcriptase found?

A

in retroviruses such as HIV

46
Q

where are restriction enzyme found? and what can they do?

A

bacteria and archaea
can cut up the viral dna to prevent the virus from making copies of itself
PROKARKOTIC DNA IS PROTECTED FROM THI =S ACTION as they are methylated at the recognition sites

47
Q

what type of “cuts” can restriction enzyme make?

A

sticky ends
blunt ends

48
Q

ligase enzyme function

A

catalyse the condensation reaction that joins the sugar groups and phosphate groups
responsible for joining together the nucleotides of the DNA fragment with the nucleotides of the plasmid by catalysing the formation of phosphodiester bonds.

49
Q

how is insulin produced fro GM bacteria?

A

-mRNA is obtained from human islet of langerhans
-reverse transcriptase used to catalyse cDNA, DNA polymerase is added to produce a double-stranded gene
- ligase enzyme can help to insert the gene into a plasmid producing a recombinant plasmid

50
Q

define gene therapy

A

mechanism by which genetic diseases are treated by masking the effect of faulty alleles by inserting a functional version of the allele, if the inserted allele is expressed, then symptoms associated with the disorder will disappear

51
Q

what is somatic cell gene therapy?

A

gene therapy by inserting functional alleles into body cells, affect only certain types of cells, alteration made to the patient are not inherited by the offspring

52
Q

what is germ line gene therapy?

A

gene therapy by inserting functional allele into gametes

53
Q

when can somatic cell gene therapy used?

A

cystic fibrosis - functioning allele for this gene can be put into specific cells so they can make the protein

54
Q

what can cause cystic fibrosis?

A

lack of functioning CFTR gene
alleles can be packaged within small spheres of lipid bilayer (LIPOSOMES)
if these are placed into an aerosol inhaler or sprayed into nose, some will pass through the plasma membrane of cells lining the respiratory tract, or they may even enter in the nucleus so they might allow the expression of CFTR protein.

55
Q

why does this therapy have to be repeated at very short intervals?

A

because epithelial cells are replaced every 10-14 days

56
Q

can GM viruses be used as vector? suggest some potential problems?

A

yes,
they might provoke an immune or inflammatory response
-patient may become immune to the virus making subsequent deliveries difficult
- virus may insert the allele in a location that disrupts a gene involved in regulating cell division
- virus may insert the allele in a location that disrupts the regulation pf the expression of other genes

57
Q

germ line therapy function

A

alter the genome of gamete, all the cells of the individual and offspring will inherit the foreign allele, descendents cannot give consent, therefore considered ethically impermissible

58
Q

proteome

A

proteins an organism can produce

Biology (18 decks)

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