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Genetics > Lesson 10 Recombinant DNA Technology > Flashcards

Lesson 10 Recombinant DNA Technology Flashcards

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1
Q

Recombinant DNA technology

A

Involves the use of in vitro molecular techniques to connect different pieces of DNA

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2
Q

biotechnology

A

Technologies that involve the use of living organisms, or products from living organisms, as a way to benefit humans

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3
Q

Transgenic organism

A

When an organism is given DNA from another organism using such techniques

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4
Q

gene cloning: two strategies

A
  1. Introducing a gene into a vector, and then propagating the vector in living cells
  2. Using the polymerase chain reaction (PCR) to make many copies of a gene in a test tube
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5
Q

vector

A

A segment of DNA that can replicate and produce many identical copies of the inserted gene.

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6
Q

When does vector replication occur?

A

When it is found within a living host cell

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7
Q

From what two natural resources were vectors used in gene cloning derived?

A
  1. Some vectors are plasmids

2. Viral vectors

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8
Q

Plasmid

A

Small circular pieces of DNA. Derived from R factors and carry antibiotic resistance genes. Contain origin of replication that is recognized by the replication enzymes of the host cell.

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9
Q

Viral vector

A

When a chromosomal gene is inserted into a viral genome, the gene will be replicated when the viral DNA is replicated.

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10
Q

Restriction endonuclease or restriction enzymes

A

Used to extract a gene from its location within a chromosome, cut DNA

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11
Q

How do restriction enzymes work?

A

They bind to a specific base sequence and then cleave the DNA backbone at two defined locations, one in each strand.

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12
Q

What do restriction enzymes digest DNA into?

A

Fragments with “sticky ends”

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13
Q

What are sticky ends?

A

The ends of these DNA fragments will hydrogen bond to each other because of their complementary sequences. The complementary sequences promote interactions between two different pieces of DNA.

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14
Q

How is hydrogen bonding between sticky ends only a temporary interaction?

A

It involves only a few hydrogen bonds between complementary bases.

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15
Q

How do you set up a permanent connection between two DNA fragments?

A

The sugar-phosphate backbones within the DNA strands must be covalently linked together by an enzyme known as DNA ligase.

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16
Q

DNA ligase

A

Covalently links sugar-phosphate backbones within DNA strands.

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17
Q

Steps to cloning a gene

A
  1. Chromosomal DNA is isolated and digested with a restriction enzyme
  2. A plasmid is cut at one site with the same restriction enzyme
  3. The digested chromosomal DNA and plasmid DNA are mixed together and incubated under conditions that will promote the binding of complementary sticky ends.
  4. DNA ligase is then added to catalyze the covalent linkage between DNA fragments
  5. After libation, DNA is introduced into bacterial cells that have been treated with agents that make them permeable to DNA molecules.
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18
Q

Hybrid vector

A

Vector containing a piece of chromosomal DNA

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19
Q

transformation

A

When a plasmid vector is used to introduce DNA into bacterial cells that have been treated with agents that make them permeable to DNA molecules

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20
Q

transfection

A

When viral vector is introduced into host cell.

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21
Q

Why are colors of colonies different that contain a recircularized vector versus a hybrid vector?

A

Blue colonies still have an intact beta-galactosidase gene; white colonies have an inactivated beta-galactosidase gene because of the insertion of a piece of chromosomal DNA

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22
Q

What two events lead to the amplification of the cloned gene?

A
  1. Bacterial host cell replicates the hybrid vector to produce many copies per cell
  2. Bacterial cells divide approximately every thirty minutes, producing a very large population of cells
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23
Q

Experiment 18A: First Gene-cloning experiment

hypothesis

A

A piece of DNA carrying a gene can be inserted into a plasmid vector using recombinant DNA techniques. If this recombinant plasmid is introduced into a bacterial host cell, it will be replicated and transmitted to daughter cells, producing many copies of the recombinant plasmid.

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24
Q

Experiment 18A: First Gene-cloning experiment

Starting materials

A

Three different strains of E. coli; one strain that did not carry any plasmid and two strains that carried pSC101 or pSC102.

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25
Q

Experiment 18A: First Gene-cloning experiment

protocol

A
  1. Isolate and purify the two types of plasmid DNA
  2. Grow the bacterial cells containing the plasmids
  3. Break open the cells.
  4. Isolate each type of plasmid DNA by density gradient centrifugation
  5. DIgest the plasmid DNAs with EcoRI
  6. Mix together the two samples
  7. Add DNA ligase
  8. Grow E. coli strain that does not carry a plasmid. Treat the cells with CaCl2 to make then permeable to DNA.
  9. Add lighted DNA samples to bacterial cells. Most cells do not take up a plasmid but occasionally one will.
  10. Plate the cells on growth media containing both tetracycline and kanamycin. Grow overnight to allow growth of visible bacterial colonies.
  11. Pick four colonies from the plates. The plasmid found in these colonies is designated in pSC105. Grow the colonies in liquid culture containing radiolabeled deoxyribonucleotides.
  12. To isolate the radiolabeled plasmid DNA, break open the cells and subject the DNA to cesium chloride density gradient centrifugation.
  13. As a control, subject pSC101 and pSC102 to the same procedure.
  14. Digest plasma DNAs with EcoRI, and subject them to gel electrophoresis
26
Q

EcoRI

A

Restriction enzyme that produces sticky ends

27
Q

Experiment 18A: First Gene-cloning experiment

Interpreting the data

A

You had two data sets. One was density from centrifuges fractions. The other was electrophoresis. In the fraction density, we saw that pSC105 had a different density than just the combination of 101 and 102, so it was recombined. Electrophoresis show that pSC105 ended up being pSC101 with just one other fragment from 102 that made the thing resistant to tetracycline and kanamycin. A recombinant molecule was created and put into E coli

28
Q

Reverse transcriptase

A

Enzyme, typically found in viruses, that can use an RNA template to make a copy of DNA

29
Q

cDNA

A

A copy of DNA made from RNA. Made with reverse transcriptase.

30
Q

cDNA synthesis: what is a poly-dT primer

A

Annals to the 3’ end of mRNAs

31
Q

cDNA synthesis: role of reverse transcriptase

A

Add reverse transcriptase + dNTPs to synthesize a complementary DNA strand

32
Q

cDNA synthesis: RNaseH role

A

RNaseH digests the mRNA into short pieces that are used as primers by DNA polymerase to synthesize the second DNA strand

33
Q

Restriction mapping

A

Location of restriction sites within a segment of DNA

34
Q

How is restriction mapping done?

A
  1. DNA is isolated and purified from host cells
  2. Samples of purified DNA are then placed in separate test tubes that contain a single restriction enzyme or a combo of different enzymes
  3. DNA is incubated with the restriction enzymes to allow sufficient time for digestion to take place
  4. DNA fragments are separated by gel electrophoresis
    5.
35
Q

Template DNA

A

The starting sample of DNA in polymerase chain reaction

36
Q

Goal of polymerase chain reaction (PCR)

A

Make many copies of the template DNA. Done by sequential rounds of DNA replication using Taq polymerase and a pair of oligonucleotide primers that are complementary to the region flanking the DNA that will be amplified. The two primers bind to the target sequence and amplify the region.

37
Q

PCR procedure

A
  1. Replica of master plate is blotted onto a nitrocellulose filter
  2. Filter is treated with SDS to permeabilize the bacteria and the DNA is fixed to the fiber
  3. Filter is then treated with sodium hydroxide to separate the DNA into single strands
  4. Radioactively labeled probes are added
  5. Probes are single-stranded DNA with base sequences complementary to that of the gene of interest
  6. Probes hybridize with the desired gene from the bacterial cell DNA
  7. Filter is washed to remove unbound probe and then exposed to X ray film.
  8. Developed film is compared to the replica of the master plate to identify bacterial colonies carrying the gene of interest
38
Q

PCR semispecific experiment

A

Primers recognize a repetitive DNA sequence that is found at several sites within the genome. When chromosomal DNA is used as a template, this will amplify different DNA fragments.

39
Q

PCR nonspecific experiment

A

A mixture of short PCR primers with many different random sequences are used. These primers will annual randomly throughout the genome and amplify most of the chromosomal DNA.

40
Q

DNA library

A

Collection of hybrid vectors

41
Q

Genomic library

A

Starting material of DNA library is chromosomal

42
Q

cDNA library

A

Starting material is from a collection of cDNA

43
Q

Colony hybridization

A

Goal: identify member(s) of library that have an insert that carries a gene of interest
A probe that is complementary to the globin gen will identify colonies that contain a hybrid vector in which the insert contains the rat globin gene

44
Q

Southern blotting

A

Used to detect a particular gene sequence within a mixture of many chromosomal DNA fragments.

45
Q

Southern blotting procedurer

A
  1. Chromosomal DNA is isolated and digested with a restriction enzyme
  2. Restriction enzyme cuts DNA at many different sites; results in thousands of DNA pieces
  3. Pieces loaded onto a gel that separates then according to their size
  4. DNA pieces in gel are dentures by soaking the gel in a NaOH solution and then the DNA is transferred to a nitrocellulose filter
  5. Determine if any of these unlabeled fragments from the chromosomal DNA contains sequence complementary to the probe, which must be labeled
  6. submerge filter into solution containing the radiolabeled probe
  7. If fragment and probe complementary, will h bond.
  8. Wash away unbound probe
  9. expose filter to x ray film; locations where radiolabeled probe has bound will appear as dark bands on the x ray film
46
Q

Variables in southern blotting procedure

A

Temperature and ionic strength of the hybridization and wash steps

47
Q

High stringency

A

high temp; low salt concentrations, probe DNA and chromosomal fragment must be very complementary - nearly a perfect match.

48
Q

Low stringency

A

Low tem; ionic strength is higher; DNA sequences that are not perfectly complementary may hybridized to the probe. Detect homologous genes

49
Q

Northern blotting

A

Like southern blotting, but detects RNA, not DNA

50
Q

Western blotting

A

Used to detect a particular protein

51
Q

Western blotting procedure

A
  1. Proteins extracted from living cells and loaded onto a gel that separates proteins on the basis of their molecular weights.
  2. Proteins within the gel are blotted onto a nitrocellulose filter and then exposed to a primary antibody that is known to recognize the protein of interest
  3. A secondary antibody, which is labeled, then binds to the primary antibody and provides a way to detect the band where the primary antibody has bound
52
Q

Gel retardation essay (band shift assay)

A

Can determine if a protein is bound to a fragment of DNA. Basis of technique: binding of a protein to a DNA fragment will regard the ability of the DNA fragment to move within a polyuacrylamide or agarose gel. The complex has a higher mass.

53
Q

DNA footprinting

A

Identifies one or more regions of DNA that interact with a DNA-binding protein. Involves the interactions between DNA, DNA-binding proteins, and agents that can chemically react with DNA.
Key point: DNaseI will not cleave the DNA in the region where RNA polymerase holoenzyme is bound. Bands are missing from the ladder where DNAseI cleavage does not occur.

54
Q

DNA sequencing

A

Technique used to determine the base sequence within a strand of DNA.

55
Q

Two methods for DNA sequencing

A

dideoxy sequencing and in vitro site-directed mutagenesis

56
Q

dideoxy sequencing

A

Uses synthetic nucleotides called dideoxynucleotides. When these nucleotides are incorporated into a growing DNA strand, they stop further growth.

57
Q

DNA dideoxy sequencing protocol

A
  1. Sample containing many copies of the ss DNA is mixed with primers that will bind to the primer-annealing site.
  2. Add four types of deoxyribonucleotides and DNA polymerase to annealed DNA fragments. Mixture divided into four separate tubes.
  3. Each of the four tubes also has a low conch. Of a different dideoxyribonucleoside.
  4. incubate to let DNA polymerase work
  5. DNA strands of different lengths will be made, depending on where bases caused chain termination
  6. Separate according to length by running them through gel
  7. Can detect because of radiolabeled deoxyribonucleotides. Visualize in x ray.
58
Q

How does dideoxyribonucleotide stop nucleotide attachment?

A

It has a hydrogen at 3’ instead of an OH group.

59
Q

How does sequencing ladder help us determine sequence?

A

We iknow which dideoxyribonucleotide was added to each tube, so we know which base is at the very end of a strand separated on gel. We can read DNA sequence by reading which base is at the end of every DNA strand separated on this gel and we can match the sequence with the length of the strand. Reading base sequence from bottom to top allows us to determine sequence.

60
Q

In vitro site-directed mutagenesis

A

Allows a researcher to direct a mutation to a specific site within a cloned DNA segment. DNA sequence can be altered in a specific way.

61
Q

In vitro site-directed mutagenesis procedure

A
  1. Oligonucleotide primer is allowed to hybridized or annual to the template DNA
    Most of primer sequence is complementary to site in the DNA where the mutation is to be made
    primer contains a region of “mismatch” where the primer and template DNA are not complementary. Mutation occurs in mismatched region.
    DNA is introduced into living cell, where DNA mismatch is repaired. It will either return to normal or result in a mutant.

Genetics (12 decks)

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