Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

BIO 2 > RECOMBINANT DNA TECHNOLOGY (LECTURE 12) > Flashcards

RECOMBINANT DNA TECHNOLOGY (LECTURE 12) Flashcards

You may prefer our related Brainscape-certified flashcards:
Biology 101 flashcards
1
Q

What does PCR stand for?

A

Polymerase chain reaction.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is polymerase chain reaction (PCR)?

A

It is the quick amplification of DNA, more specifically the amplification of specific genes.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

How long does polymerase chain reaction (PCR) take?

A

A few hours.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

How is polymerase chain reaction (PCR) so specific?

A

Primers are designed to target a specific DNA sequence within an entire genome.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Does polymerase chain reaction (PCR) require lots of DNA?

A

No, only a small amount of DNA is required.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Name an application of polymerase chain reaction (PCR).

A

COVID testing.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What must be combined in a tube for polymerase chain reaction (PCR) to proceed? (4)

A

DNA containing specific sequence we wish to amplify, primers, nucleotides (dNTPs) and DNA polymerase (Taq).

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is particular about the primers used in polymerase chain reaction (PCR)?

A

The primers are short pieces of single stranded DNA that are complementary to either end of the gene we wish to amplify.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What is particular about the DNA polymerase (Taq) used in polymerase chain reaction (PCR)?

A

It is a thermostable DNA polymerase.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

List the 3 steps to polymerase chain reaction?

A

(1) Denaturation (2) Annealing (3) Extension / polymerization.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Describe the denaturation step of PCR.

A

Tube is heated to 95 Celsius to denature DNA (unzipping the DNA).

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Describe the annealing step of PCR.

A

Cool the tube to 55 degree Celsius to allow the primers to bind to the DNA.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Describe the extension / polymerization step of PCR.

A

Tube is heated to 72 degrees Celsius for Taq DNA polymerase to synthesize complementary DNA.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Are the 3 steps to PCR repeated?

A

Yes! The cycle is repeated over and over for exponential amplification.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

After how many cycles of PCR is the desired fragment of DNA finally isolated?

A

After the 3rd cycle, the desired fragment of DNA has been isolated.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Is there any point to performing more cycles of PCR after having isolated the desired DNA fragment?

A

Yes - every subsequent cycle will further amplify / replicate the desired DNA fragment.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What does it mean to clone a gene?

A

It means to make multiple copies of a gene.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What are the 2 primary purposes for cloning a gene?

A

To produce a protein or to produce copies of a gene to insert it somewhere else.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

What are examples of proteins for which we would want to clone a gene? (2)

A

Insulin, growth hormone.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

What is an example of gene we would want to clone in order to insert it into another organism?

A

Genes for pest resistance to insert into a plant.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

What primary tools are required for cloning? (3)

A

(1) Plasmid (2) Gene of interest (3) Restriction endonucleases.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

What is the source for a gene of interest for cloning?

A

Either DNA or cDNA.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

How can we obtain large quantities of a gene of interest for cloning?

A

By amplifying that gene using PCR.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

What do restriction endonucleases do in cloning?

A

The hydrolyze phosphodiester bonds and cut DNA at specific restriction sites - both strands are cut to produce overhangs called “sticky-ends”.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

What are the 5 steps to cloning?

A

(1) Isolate plasmid from bacteria and isolate DNA containing gene of interest. (2) Amplify gene of interest using PCR. (3) Cut plasmid and amplified gene of interest with same restriction enzyme. (4) Mix cut DNA together and add ligase to produce recombinant plasmids. (5) Transform bacteria with recombinant plasmids and select for transformed bacteria using antibiotic resistance.

26
Q

After the gene of interest is amplified during cloning (step 2), why do the replicated genes suddenly have the same restriction sites used to cut the plasmid?

A

During the amplification of the gene of interest (PCR), the primers used are made to contain the same restriction site(s) as the plasmid does. These sites are thus added to the ends newly synthesized copies of the target sequence.

27
Q

Why do we cut the plasmid and amplified gene of interest (cloning step 3) with the same restriction enzyme?

A

By cutting them with the same restriction enzyme, they will have complementary sticky ends.

28
Q

Why do we add ligase along with the cut plasmid and cut gene of interest in cloning step 4?

A

So that after the complementary sticky ends of the plasmid DNA and gene of interest base pair together, the DNA ligase will “glue” the strands together.

29
Q

How do we select for transformed bacteria in step 5 of cloning?

A

Plate the cells on an agar plate containing antibiotics (such as ampicillin) and thus only cells with recombinant plasmid (containing genes to resist antibiotics) will grow on antibiotic plate.

30
Q

Define colony.

A

It is many cells that have risen from the same cell.

31
Q

What are the 2 sources of DNA for cloning?

A

(1) Genomic library and (2) cDNA (complementary DNA) library.

32
Q

What is a genome?

A

The total DNA in a cell.

33
Q

What is the genomic library?

A

It is a collection of DNA fragments of the entire genome produced by restriction digest. Each fragment is cloned into a plasmid.

34
Q

How is the cDNA (complementary DNA) library made? (4)

A

mRNA’s are isolated from a cell. Reverse transcriptase enzyme is used to make a complementary strand. DNA polymerase synthesizes the second DNA strand to make a double stranded cDNA molecule. cDNA molecules cloned onto plasmids.

35
Q

What is the primary difference between a genomic library and a cDNA library?

A

cDNA library only includes the transcribed coding sequences of a genome.

36
Q

Will cDNA have introns?

A

No.

37
Q

What is the primary problem with cloning?

A

Eukaryotic genes are not always expressed well in prokaryotic cells.

38
Q

What are the 3 reasons that eukaryotic genes are not always expressed well in prokaryotic cells.

A

(1) Different regulatory elements are present in prokaryotes. (2) Introns cannot be spliced out in bacterial cells. (3) Proteins are not post-translationally modified in bacteria.

39
Q

Eukaryotic genes are not always expressed well in prokaryotic cells because different regulatory elements are present in prokaryotes (reason 1). What is a solution to this cloning problem?

A

Put the eukaryotic gene under control of prokaryotic promoter.

40
Q

Eukaryotic genes are not always expressed well in prokaryotic cells because introns cannot be spliced out in bacterial cells (reason 2). What is a solution to this cloning problem?

A

Use cDNA as the source of DNA.

41
Q

Eukaryotic genes are not always expressed well in prokaryotic cells because proteins are not post-translationally modified in bacteria (reason 3). What is a solution to this problem?

A

Express the cloned genes in eukaryotic cells (simpler ones that behave like bacteria, such as yeast).

42
Q

When genomic DNA from different individuals is digested with the same restriction enzymes, the pattern of DNA fragments produced is different. Why?

A

This difference is caused by single nucleotide polymorphisms (SNPs) scattered throughout the genome. SNPs cause RFLPs (restriction fragment length polymorphisms) between individuals.

43
Q

What is restriction fragment analysis?

A

The analysis of differing restriction fragment length between individuals after having cut their DNA with the same restriction enzymes.

44
Q

What are the 2 primary uses for restriction fragment analysis?

A

DNA fingerprinting and distinguishing between normal and disease allele.

45
Q

Explain 2 ways in which DNA fingerprinting is applied?

A

DNA fingerprinting to match a suspect’s DNA to DNA left at a crime scene, also to determine paternity.

46
Q

Why can restriction fragment analysis be used to determine paternity if a son and his father are 2 different individuals with different SNPs?

A

Because RFLPs are inherited in a Mendelian way.

47
Q

Explain how restriction fragment analysis can still be used to distinguish between normal and disease alleles when there aren’t any SNPs in that allele’s genes?

A

Even SNPs next to a gene can be used to differentiate between a normal and a disease causing allele.

48
Q

What is the most basic / common technique for sequencing DNA?

A

The dideoxy chain termination method.

49
Q

What ingredients are required to sequence DNA using the dideoxy chain termination method? (5)

A

DNA to sequence, primer, DNA polymerase, deoxyribonucleotides (dNTPs) and dideoxyribonucleotides (ddNTPs).

50
Q

What is particular about DNA to use that we wish to sequence using the dideoxy chain termination method? (2)

A

The DNA used is a template of the result of the sequencing. The DNA is also denatured for sequencing to proceed.

51
Q

ddNTPs are modified ____ that act as ____ ____.

A

ddNTPs are modified dNTPs that act as chain terminators.

52
Q

How many types of ddNTPs are there? How are they told apart?

A

Each of the 4 ddNTPs is tagged with a different coloured fluorescent tag.

53
Q

How is a ddNTP different from a dNTP? (2)

A

The hydroxyl group is replaced by a hydrogen on the 3’ carbon. Thus no nucleotides can be added to the chain of nucleotides following the addition of a ddNTP.

54
Q

What are the steps of the dideoxy chain termination method? (3)

A

(1) Mix all 5 ingredients together. (2) DNA polymerase synthesizes complementary DNA strands to the DNA templates. (3) DNA fragments are separated by size.

55
Q

When will the DNA synthesis of a complementary strand be terminated in step 2 of the dideoxy chain termination method?

A

As soon as a ddNTP is incorporated into the complementary strand.

56
Q

How will the fragments produced in step 2 of the dideoxy chain termination method be differentiated?

A

DNA fragments will be fluorescently labeled and the colour of fluorescence depends on which ddNTP terminated the strand.

57
Q

Will the DNA fragments produced in step 2 of the dideoxy chain termination method be of the same size?

A

No, there will be fragments of every possible length.

58
Q

What ddNTPs correspond to which NTPs?

A

ddATP corresponds to adenine, ddCTP corresponds to cytosine, ddGTP corresponds to guanine and ddTTP corresponds to thymine.

59
Q

How can we separate amino acids by size in step 3 of the dideoxy chain termination method?

A

By gel electrophoresis.

60
Q

Explain how separating DNA fragments by size in step 3 of the dideoxy chain termination method gives the order of bases in the original DNA sample.

A

As DNA fragments move through the gel they pass through a fluorescence detector. The colour of the fluorescence detected tells you which base is in each position. Fragments move past the detector from smallest to largest and the order of fluorescence detected tells you the sequence of bases in the DNA.

BIO 2 (14 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions