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biotechnology Flashcards

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1
Q

what is biotechnology?

A

technology that harnesses biological processes and develops products that can help improve our lives

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2
Q

what is modern biotechnology?

A

it combines recent discoveries in molecular biology and genetics with more traditional biotechnologies such as selective breeding and hybridization

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3
Q

what are tools present in modern biotechnology?

A

-restriction enzymes
-recombinant DNA
-electrophoresis
-vectors (plasmids, phages)
-PCR

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4
Q

what are restriction endonucleases (restriction enzymes)?

A

-enzymes that act like molecular scissors that recognize a specific DNA sequence and cleave the DNA
-original function in bacteria is to degrade the DNA of bacteriophage viruses, thus restricting invasion viruses in the bacteria
-can restrict virus from infecting them by producing enzymes that recognize specific DNA sequences and cleave them

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5
Q

which bonds do restriction enzymes specifically attack?

A

they cut the phosphodiester bonds of the phosphate sugar backbone of DNA

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6
Q

How come the bacteria’s own DNA does not get cut by restriction enzymes?

A

Bacteria have a specific methylsse enzyme that recognizes the restriction site of the bacterial genome and adds a methyl group

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7
Q

which type of restriction enzyme am I:
recognizes the sequence but cuts randomly in q region of DNA which is not the recognition region (not useful in biotechnology)

A

type I and III

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8
Q

which type of restriction enzyme am I:
recognizes a specific sequence (4 to 12 bpP and cuts a defined region of DNA within the recognition region (useful in biotechnology)

A

type II

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9
Q

what are stricky ends or everhangs?

A

if the 2 cut sites are not directly opposite each other, the regions in front of the cut sites are free and easily linkable

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10
Q

what are blunt ends?

A

if the 2 cut sites are directly opposite each other, the remaining fragments are flat

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11
Q

what am I:
technique that distinguishes individuals, populations, or species by using the intrinsic difference in the locations of their restriction enzyme sites

A

restriction fragment length polymorphism (RFLP)

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12
Q

what is gel electrophoresis and what does it do?

A

-separates and purifies DNA fragments
-consists of a gel, which is made of either agarose or polyacrylamide, in a bath of buffer solution containing ions with a negative and positive electrodes at both end of the gel
-DNA is loaded on the negative end in small rectangular shape and cut in the gel
-since DNA is negatively charged, due to PO4 groups in DNA backbone, so when a current is applied to the gel, the DNA will migrate toward the positive pole

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13
Q

what is recombinant DNA and what does it do?

A

-restriction enzymes can be used with ligase enzyme to recombine different DNA together
-permits to precisely cut at known sequence of DNA
-once the DNA is cut, you can insert another fragment of DNA

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14
Q

what is DNA ligase and what does it do (recombinant DNA)?

A

-enzyme that binds 2 DNA fragments together (blunt or sticky ends)
-creates a covalent bond between 5’ PO4 and 3’ OH end of fragments
-resulting fragment is called recombinant DNA

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15
Q

what am I:
-DNA molecule which is sued as a vehicle that carries foreign genetic material which is artificially prepared into another cell
-can be bacteriophage or plasmid

A

vector

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16
Q

what am I:
-virus that infects bacteria by injecting their DNA

A

bacteriophage

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17
Q

what am I:
extra piece of circular DNA naturally occurring in some bacteria

A

plasmid

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18
Q

what are the characteristics of a plasmid vector?

A

-they have an origin of replication so that DNA is replicated to the host cell
-they have a restriction site sot that the vector can be open in one place to insert the foreign DNA
-can be used with differential and selectable markers to determine if host cells contain the vector complex with the foreign DNA

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19
Q

what are the 3 steps to using vectors in biotechnology?

A

1) digestion
2) ligation
3) transformation

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20
Q

which step am I:
the vector and foreign DNA are cut with the same restriction enzyme

A

digestion

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21
Q

which step am I:
foreign DNA is inserted in the plasmid and the DNA ligase is used to fuse the DNA together

A

ligation

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22
Q

which step am I:
the plasmid is introduced in a bacteria (heat shock of electroporation)

Study These Flashcards
A

transformation

23
Q

what is one technique used to know which bacteria is transformed?

Study These Flashcards
A

-genes that have a restriction site are used so that the foreign DNA can be inserted
-this will disrupt the differential maker gene and have a visual effect when bacteria is cultured

24
Q

what is PCR?

Study These Flashcards
A

technique that permits the amplification and replication of specific region of the DNA without the need of a host cell

25
what are the 5 elements you need for PCR?
1) primers (DNA) 2) free nucleotides 3) DNA polymerase (Taq) 4) reaction buffer 5) thermocycler machine
26
what are primers (PCR)?
-20-25 nucleotides long that are complementary to the DNA adjacent to the gene that is to be replicated -no primase enzyme needed
27
what is Taq polymerase?
-comes from bacteria -used in PCR because human enzyme would be denatured during the process
28
what does the thermocycler machine do during PCR?
raises and lowers the temperature of the samples in discrete, pre-programmed steps
29
which step of PCR am I: DNA samples are heated (95) and denatured so the 2 strands separate (the heat breaks the h-bonds between the base pairs)
denaturation
30
which step of PCR am I: the temperature is cooled (45-54) and the primers anneal to the specific region
annealing
31
which step of PCR am I: Taq polymerase extends the DNA (72) thus giving new copies of the DNA (primers are part of the amplified sequence)
extension
32
what is reverse transcription PCR?
-reverse transcriptase is an enzyme found in retrovirus to copy their viral RNA into the genome of the cell host -used in biotechnology to transcribe the m-RNA into coding DNA (no introns)
33
what is DNA cloning?
to make a genetically exact copy out of something
34
what is a DNA library?
collection of DNA fragments that is stored and propagated in a population of micro-organisms through the process of molecular cloning
35
what are the 2 types of DNA libraries?
genomic DNA library cDNA library
36
which type of DNA library am I: -represents the entire genome cut and inserted into plasmids in bacteria -less useful in eukaryotic cells since DNA contains introns thus the gene cannot be expressed by bacteria
DNA genomic library
37
how does bacteria multiplication work with a DNA genomic library?
-DNA is digested with restriction enzymes -DNA fragment is ligated in a plasmid -bacteria is transformed -bacteria multiply (cloning of the plasmid)
38
what is the utility of DNA genomic library?
-used for sequencing applications -played important role in whole genome sequencing -clones from genomic libraries amplify the DNA fragment (technique developed for sequencing in 1980s, obsolete now)
39
how to find a gene in the DNA genomic library?
-southern blot A technique: DNA fragments are separated by gel electrophoresis, denatured into single-stranded DNA, and then "blotted" onto a sheet of filter paper; the filter is then incubated with a probe to locate DNA sequences of interest
40
which type of DNA library am I: -collection of only the genes that are encoded into proteins by an organism -RT PRC used to make this library
cDNA library
41
42
43
what does DNA fingerprinting use?
-restriction fragment length polymorphisms: -fragments genome by cutting with restriction enzymes -DNA fragments are migrated on an electrophoresis gel to separate the DNA by size band pattern is compared needs a lot of DNA (out-dated method)
44
what is sequencing?
the pocoess of determining the order of nucleotides in DNA
45
46
how does the Sanger method work?
-some of the nucleotides do not have OH (thus no other nucleotide can be added after which terminates the sequence) -these special nucleotides = ddNTP in manual method ddNTP were radioactive: -when PCR was done each ends stopped at known radioactive ddNTP -to know the DNA sequence you needed to migrate the amplified DNA on a gel and expose the gel to a film to reveal where the radioactive ddNTP is modern method uses fluorescent ddNTP
47
what is the next generation sequencing method and how does it work?
-no electrophoresis gel -DNA is cut and attached to a solid surface -DNA is copied by PCR to turn one molecule of DNA into a cluster of identical DNA pieces, hence making a denser spot (easier for computer to image) -PRC is made with special fluorescent nucleotide -once fluorescent base binds to the DNA, a picture of the flow cell will be taken (since each nucleotide has a distinct color, we can find out which one it is) -dye is washed off and a second base binds to the cluster cycle is repeated 100-200 times
48
why can't restriction enzymes type II be used to modify the genome in a specific way?
because they generally recognize 4-12 bp and cut the DNA on average once every 4096 to 65536 base pair, while for genome engineering we needed enzymes that recognize sequences 16-18 bp to create a unique targeted cut in genome
49
what is CRISPR?
important component of bacterial immune system that allows bacteria to remember and destroy phages
50
what are the 2 parts to CRISPR?
1) cas9 enzyme: unwinds and cuts DNA 2) guide RNA: recognizes the sequence of DNA to be edited in the genome
51
what does CRISPR stand for?
clustered regularly interspaced short palindromic repeats
52
how does CRISPR/Cas9 work?
1) foreign viral DNA enters cell, cas complex recognizes it and splits it into small fragments 2) one of the virus fragment is added to CRISPR array in the bacterial genome 3) CRISPR array is transcribed into a long RNA 4) CRISPR array is then cut in a small piece representing a specific virus 5) there rRNAs direct the Cas complex to foreign DNA according to the specificity of the sequence 6) if the specific DNA is recognized by the RNA, then it is cleaved, thus eliminating the virus DNA
53
what can CRISPR CAS9 be used to do?
-delete a portion of a gene to inactivate it (gene silencing) -insert a portion of DNA in a gene -correct a portion of gene using a donor DNA as template

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