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Genetics Test 5 > 38 Biotechnology > Flashcards

38 Biotechnology Flashcards

1
Q

recombinant DNA

A

the product of joining DNA sequences from different sources together

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2
Q

what is a key factor in creation of recombinant DNA?

A

the ability to reproducibly cut DNA at a specific site to have a predictable result and ability to replicate

accomplished by restriction enzymes

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3
Q

restriction enzymes

A

endonuclease that recognizes a specific DNA sequence and cleaves dsDNA at that sequence

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4
Q

palindrome

A

reads the same 5’ to 3’ on either strand for a segment of DNA

recognition sites for restriction enzymes

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5
Q

Is GATTAG a palindrome?

A

no - second strand is CTAATG

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6
Q

Is GAATTC a palindrome?

A

yes - second strand is CTTAAG

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7
Q

sticky ends

A

bases at the end sequences but by restriction enzymes that are not paired

will want to base pair or “stick” to complementary sequences

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8
Q

steps to make recombinant DNA

A
  • cut DNA from two sources with same restriction enzyme
  • mix products
  • some pieces from one source will join with pieces from the other source at their sticky end
  • restores complementary pairing but DNA ligases is added to seal the nick in the sugar phosphate backbone
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9
Q

vector

A

carrier DNA molecule capable of independent replication into which a DNA fragment can be cloned

purpose is to carry foreign DNA into the cell

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10
Q

characteristics of an ideal cloning vector

A
  • origin of replication
  • selectable/insertional markers allowing identification
  • single cleavage site for each restriction enzyme
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11
Q

examples of vectors

A

plasmids
lambda phage
retroviruses

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12
Q

ligation experiment

A

conducted to join foreign DNA to vector

foreign DNA and vector are both cut by same restriction enzyme, they are mixed and ligase is added - some recombinant molecules should form

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13
Q

insertional inactivation

A

the inserted DNA’s inactivation of a gene in the vector when it inserts into that genes - allows cells with the recombinant DNA to be identified

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14
Q

transformation experiment

A

conducted to allow cells to take up products from ligation experiment

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15
Q

ligation and transformation experiments are constructed to allow….

A

identification of different cell types including…

  • cells with no uptake
  • cells that took up the original vector
  • cells that took up the recombinant plasmid
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16
Q

plasmid

A

double stranded circular DNA that replicates independently of the bacterial chromosome

have features useful for cloning

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17
Q

polylinker

A

multiple cloning site

segment of DNA that has a lot of recognitions sites for restriction enzymes so that there is flexibility of enzyme choice depending on what enzyme recognition sites are present in the foreign DNA

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18
Q

example of interstitial inactivation

A

insert a gene into the polylinker of the tet gene inactivates the tet resistance gene

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19
Q

selectable marker

A

all bacteria successfully transformed contain the trait coded for in this region - ex may have amp resistance this allows selection of only transformed colonies

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20
Q

ori site

A

origin of replication

allows the plasma to replicate in the cell independently of the bacterial chromosome

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21
Q

features of plasmids useful to cloning

A

multiple cloning site
insertional marker
selectable marker
origin of replication

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22
Q

insertional marker

A

if foreign DNA is successfully inserted, insertional inactivation occurs

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23
Q

What three chemicals are added to the media in blue and white screening

A

ampicillin
IPTG
X-gal

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24
Q

purpose of ampicillin in blue and white screening

A

the plasmid added contains an ampicillin resistance gene as its marker

only the cells who took up the plasmid will grow on the medium

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25
Q

purpose of IPTG in blue and white screening

A

IPTG is a synthetic inducer to turn on lacZ gene to produce beta galactosidase which will break down X-gal into galactose derivative and a blue pigment

only cells that did not take up the plasmid will grow blue as
cells that took up the plasmid will grow white as the plasmid inactivates the lacZ gene

26
Q

Why did scientists want to create golden rice?

A

to add provitamin A to rice to help prevent the consequences of vit A deficiency in kids

27
Q

What is rice missing to make beta carotene?

A

phytoene and lycopene

28
Q

What genes needed to be added to make golden rice?

A

PSY - converts GGPP to phytoene

CRIT - converts phytoene to lycopene

29
Q

the original PSY gene was isolated form…

A

daffodil

30
Q

CRIT is isolated from….

A

bacteria

31
Q

Why is cDNA needed?

A

mRNA can be isolated from the gene, but needs to be DNA to be inserted into the cell

cDNA is produced with the help of reverse transcriptase (a RNA dependent DNA polymerase) using mRNA as the template

32
Q

transgenic plant or animal

A

one in which a foreign or exogenous gene has been introduced into its genome altering its genetic constitution

can also occur when the gene is derived from their own genome but is altered in some way

33
Q

Ti plasmid

A

tumor inducing plasmid causing gall disease by insert itself into the plant chromosome

used as a vector after removing the tumor causing genes but not the transfer functions

34
Q

expression vectors

A

allow the inserted gene product to be produced

must contain sequences required for transcription and translation of the gene in addition to other vector characteristics - marker gene, origin of replication, promoter in proper orientation

35
Q

components of construct for insertion into rice genome

A
  1. PSY gene (has own promoter and poly a site)
  2. crtl gene (has own promoter and poly a site)
  3. endosperm specific promoters
  4. poly a signals
  5. transgenic marker
  6. LB and RB for insertion into plant genome
36
Q

construct

A

the DNA sequence that will be put into the vector

37
Q

what was used to separate DNA fragments for the construct for golden rice

A

electrophoresis

38
Q

describe electrophoresis

A

DNA fragments loaded on neg side

migrate towards positive side with smaller pieces migrating more rapidly

distance is inversely proportional to the log of the fragment size

sizes determined against a control

39
Q

blotting

A

process of transferring molecules that were previously separated to a membrane that is better able to support additional testing

40
Q

south blot

A

DNA fragment separated based on length

41
Q

northern blot

A

RNA fragments separated based on length

42
Q

western blot

A

proteins that are separated on molecular weight, isoelectric point, electric charge etc

43
Q

what was used to identify which plants had the inserted genes in the creation of golden rice?

A

southern blot

single stranded dna fragments from the gel are transferred to a nylon membrane using capillary action

44
Q

probe

A

a single stranded DNA that is a sequence we’re interested in

binds to complimentary DNA fragments allowing us to see the position of the prob

45
Q

second source of PSY for golden rice

A

maize

much higher beta-carotene production than that with daffodil gene

46
Q

Sanger sequencing reaction

A
  • normal deoxyribonucleoside triphosphate has 3’OH
  • dideoxyribonucleoside triphosphate has 3’H but can be incorporated into DNA in replication at which point replication will stop as no additional bases can be added to 3’H
  • allow replication to proceed in 4 different tubes, each with a different dideoxy base
  • detect the position each dideoxy nucleotide was incorporated into
47
Q

in sanger sequencing we read the gel from…to read the sequence from…

A

bottom to top

5’ to 3’

48
Q

genes amplified for identification

A

highly polymorphic regions where a short segment is repeated different numbers of times in different alleles

STR - short tandem repeats
VNTR - variable number of tandem repeats
microsatellite regions

49
Q

PCR

A

polymerase chain reaction

idea is to get many copies of a specific dna sequence produced automatically

50
Q

steps of PCR

A

Denature DNA by heating to 95 Celsius
each strand is template for replication
primers anneal to identify target that will be amplified
Taq polymerase adds nucleotide to 3’ end of primer.
Repeat many times

51
Q

polymerase used in PCR

A

taq polymerase
a polymerase isolated from bacteria in hot springs
all materials added to 1 tube at beginning of PCR process, therefore must be able to withstand the range of temperatures required for the reaction

52
Q

purpose of primers in PCR

A

one primer binds to each template just upstream from the sequence being amplified to allow replication

53
Q

Limitations to PCR

A
  • must know something about sequence surrounding the gene of interest to design proper primers
  • PCR reactions are easily contaminated from other DNA in the lab
  • taq polymerase does not proofread and correct errors making the error rate high
  • fragment amplified are relatively small
54
Q

electrophoresis role in amplification

A

allows comparison of DNA profiles from different individuals

55
Q

why are STRs used in amplification

A

different alleles have different numbers of copies of the repeat

they are polymorphic so there will be differences in lengths between individuals

56
Q

paternity test

A
  • isolate DNA from each individual and amplify the region using PCR, then separate with electrophoresis
  • compare the sizes of fragments of child to potential parents
57
Q

In forensics the DNA pieces must be…

A

exactly the same in the suspect sample as in the evidence sample

58
Q

In paternity testing, the DNA pieces must…

A

be half from mom and half from dad

59
Q

CODIS

A

combined DNA index system

DNA database funded by FBI

uses 20 polymorphic STR regions for forensic identification

60
Q

Where do the DNA samples in CODIS come from?

A
  • from offenders
  • from crime scenes
  • from missing persons, relatives of missing persons, and unidentified remains
  • automatically added if youre arrested for violent crime - can be requested to be purged if exonerated but if convicted will stay in database
61
Q

familial DNA testing

A

when family members are asked to submit their DNA for testing in order to find a perfect match when the suspect has a partial, very close DNA match

62
Q

innonance project

A

uses dna evidence to exonerate people who were falsely imprisoned

Genetics Test 5 (5 decks)

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