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BIOL114 Biotechnology > Gene Analysis > Flashcards

Gene Analysis Flashcards

Complete deck covering Lecture 2

1
Q

List four techniques to study gene / DNA structure / sequences

A
  1. Restriction Mapping/Restriction Fragment Length Polymorphism (RFLP)
  2. Polymerase Chain Reaction
  3. Southern Analysis
  4. DNA Sequencing
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2
Q

True or False:

Agarose gel electrophoresis separates DNA fragments by size.

A

True

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3
Q

Fill in the gaps: (Agarose gel electrophoresis)
A gel made of a polymer, such as ___________ _______, acts as a _________ _____ to separate nucleic acids on the basis of ____.

An ________ ______ is applied to separate them based on their ___________ properties. Nucleic acids carry _________ charges on their ________ groups, so travel to the _______ pole in an electric field.

An Agarose Gel is a ___ _____of interconnected agarose molecules which form _______ and _____ through which biomolecules can pass.

A

A gel made of a polymer, such as polysaccharide agarose, acts as a molecular sieve to separate nucleic acids on the basis of size.

An electrical charge is applied to separate them based on their electrochemical properties. Nucleic acids carry negative charges on their phosphate groups, so travel to the positive pole in an electric field.

An Agarose Gel is a 3D matrix of interconnected agarose molecules which form channels and pores through which biomolecules can pass.

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4
Q

Fill in the gaps: (Agarose gel electrophoresis)
___________ of the Agarose gel influences how big or small the pores are in the matrix. The gel ______ the movement of ______ molecules more than the ______ ones, separating them.

Normally in a tube there is a mixture of the DNA molecule of different sizes, plus ____ that allows us to see the content of tube, and _______ or ______, to make the solution _____ ____ into our wells.

A

Concentration of the Agarose gel influences how big or small the pores are in the matrix. The gel impedes the movement of longer molecules more than the shorter ones separating them.

Normally in a tube there is a mixture of the DNA molecule of different sizes, plus dye that allows us to see the content of tube, and glycerol or sucrose, to make the solution load well into our wells.

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5
Q

What is a molecular weight marker for DNA? How does it work?

A

A piece of DNA cut by restriction enzymes to give fragments of a known size.

They are used to estimate the size of DNA of interest by comparison.

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6
Q

Fill in the gaps: (Agarose gel electrophoresis)
To _________________________________
________________ while running the gel in the electrophoresis tank, a buffer that ___________
______________________ is used.

To allow a _____ ________ of the DNA, it is important to ______________________.

A

To prevent overheating and melting of the agarose while running the gel in the electrophoresis tank, a buffer that allows the current to pass through the agarose gel is used.

To allow a good migration of the DNA, it is important to use the right amount of buffer.

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7
Q

Fill in the gaps: (Agarose gel electrophoresis)
To make the loading easier and prevent the DNA from ___________________, add a _______ _______ containing some ______ or ______ to the DNA sample when loading the sample into the wells. This ensures it _________________.

A

To make the loading easier and prevent the DNA from diffusing into the buffer, add a loading buffer containing some sugar or glycerol to the DNA sample when loading the sample into the wells. This ensures it remains inside the well.

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8
Q

During agarose gel electrophoresis, what dye used to be the most commonly used in the gel and why?

A

Ethidium bromide

It fluoresces in UV light, allowing the DNA to be visible.

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9
Q

True or False:

GelRed is a safer alternative dye to ethidium bromide

A

True

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10
Q

True or False:

Ethidium bromide is a safer alternative dye to GelRed

A

False

GelRed is a safer alternative dye to ethidium bromide

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11
Q

What is a safer alternative dye to ethidium bromide?

A

GelRed

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12
Q

Fill in the gaps: (Agarose gel electrophoresis)
GelRed is a _____, __________ molecule visible under ___. These molecules intercalate into _____________ and make it possible to visualize it by ___________ if the gel is _______________.

This allows a _______________ corresponding to the DNA that was loaded and ran to be seen.

A

GelRed is a small fluorescent molecule visible under UV. These molecules intercalate into the double helix and make it possible to visualize it by fluorescence if the gel is irradiated with UV.

This allows a pattern of bands corresponding to the DNA that was loaded and ran to be seen.

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13
Q

True or False:

How a DNA fragment is cut depends on the combination of restriction endonucleases used

A

True

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14
Q

True or False:

How a DNA fragment is cut does not depend on the combination of restriction endonucleases used

A

False

How a DNA fragment is cut does depend on the combination of restriction endonucleases used

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15
Q

Fill in the gaps:
In agarose gel electrophoresis, if some DNA is cut using restriction endonucleases into two pieces ________________, e.g. ____ into ______, this will show up on the ______ ______ so only one of the pieces will be detected.

A

In agarose gel electrophoresis, if some DNA is cut using restriction endonucleases into two pieces of the same length, e.g. 10kb into 2 x 5kb, this will show up on the same band so only one of the pieces will be detected.

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16
Q

What can restriction fragment analysis be used for?

A

Comparing two different DNA molecules, such as two different alleles for a gene

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17
Q

In restriction fragment analysis, what is a possible consequence of a nucleotide difference in the DNA?

A

If the difference affects the recognition site of a restriction enzyme, could prevent the restriction enzyme from cutting the DNA

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18
Q

What is the name given to variations in DNA sequence among the population?

A

Polymorphisms

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19
Q

Define polymorphisms

A

Variations in DNA sequence among the population

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20
Q

What name is given to sequence changes which impact the recognition site of restriction enzymes?
R________ F________ L_____ P___________

A

Restriction fragment length polymorphisms

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21
Q

How does RFLP Analysis determine differences in alleles?

A

The pattern of bands from an electrophoresis allows detection of normal alleles and sometimes mutant alleles.

If a mutation that disrupts one of the recognition sites for a restriction endonuclease is present, the band pattern will be different.

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22
Q

Fill in the gaps: RFLP analysis for sickle-cell alleles

The normal allele for beta globin includes four recognition sites for the restriction enzyme called DdeI. Ddel cuts these, generating three fragments clearly detectable in an agarose electrophoresis. A fragment of 175bp, a fragment of 201bp and a much larger fragment are created.

The sickle-cell mutant allele for beta global includes a mutation that disrupts one of the recognition sites for Dde I (at the second cut) so only two fragments are generated. Neither of these are 201bp or 175bp.

A

The normal allele for beta globin includes four recognition sites for the restriction enzyme called DdeI. Ddel cuts these, generating three fragments clearly detectable in an agarose electrophoresis. A fragment of 175bp, a fragment of 201bp and a much larger fragment are created.

The sickle-cell mutant allele for beta global includes a mutation that disrupts one of the recognition sites for Dde I (at the second cut) so only two fragments are generated. Neither of these are 201bp or 175bp.

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23
Q

Fill in the gaps: RFLP analysis for sickle-cell alleles

The normal allele for ____ ______ includes ____ recognition sites for the restriction enzyme called _____. Cuts at those sites generates ____ fragments clearly detectable in an ________ ____________. A fragment of ___bp, a fragment of ___bp and a much larger fragment are created.

The sickle-cell mutant allele for ____ ______ includes a mutation that disrupts ___ of the recognition sites for ____ (at ___________) so only ____ fragments are generated ( ___bp and a much larger fragment).

The electrophoresis gives a different banding pattern, which highlights the mutant allele.

A

The normal allele for beta globin includes four recognition sites for the restriction enzyme called DdeI. Cuts at those sites generates three fragments clearly detectable in an agarose electrophoresis. A fragment of 175bp, a fragment of 201bp and a much larger fragment are created.

The sickle-cell mutant allele for beta globin includes a mutation that disrupts one of the recognition sites for Dde I (at the second cut) so only two fragments are generated ( 376bp and a much larger fragment).

The electrophoresis gives a different banding pattern, which highlights the mutant allele.

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24
Q

If the full genomic DNA of an organism is cut with restriction enzymes and ran through an electrophoresis analysis, what would be detected?
Why?

A

Would look like a smear, due to containing hundreds of thousands of bands, because the full genomic DNA would have so many restriction enzyme recognition sites

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25
Q

What are the three steps for a polymerase chain reaction (PCR)?

A
  1. DNA is heated to 90-95°C to separate the double strands of the DNA we are using as a template
  2. A primer specific to the sequence we wish to amplify is annealed (forms hydrogen bonds) at 50-65°C
  3. Temperature raised to 70°C to allow thermostable DNA polymerase to synthesise two new strands
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26
Q

Give an example of a thermostable DNA polymerase

A

Taq polymerase

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27
Q

List some things polymerase chain reactions (PCR) can be used for?

A 
Amplifies DNA in an exponential chain reaction (1 molecule can become 2^(30) molecules in hours)
Cloning
Species identification (meat scandals)
Disease allele identification
Forensics
Gene expression
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28
Q

Where do we get thermostable DNA polymerases?

Give a specific named example

A

They are isolated from bacteria that live in very hot places

Thermus aquaticus

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29
Q

Fill in the gaps

The development of PCR led to ____ _____ receiving the ____ Nobel Prize in ________

A

The development of PCR led to Kary Mullis receiving the 1993 Nobel Prize in Chemistry

30
Q

Fill in the gaps:
In PCR primers specific to the sequence we wish to amplify are added and allowed to anneal ( meaning they __________________________________). Two are used, ______________.

A

In PCR primers specific to the sequence we wish to amplify are added and allowed to anneal (meaning they form hydrogen bonds / base pair with the complimentary sequence on the single stranded DNA template). Two are used, one for each DNA strand.

31
Q

True or False:

In PCR the DNA is first heated to 90-95°C

A

True

32
Q

True or False:

In PCR the DNA is first heated to 50-65°C

A

False

In PCR the DNA is first heated to 90-95°C

33
Q

What temperature do thermostable polymerases usually work well at?

A

70°C

34
Q

What types of DNA can be used as a template in PCR?

A

Genomic DNA

cDNA

35
Q

True or False:

Genomic DNA can be used in PCR

A

True

36
Q

True or False:

Genomic DNA cannot be used in PCR

A

False

Genomic DNA can be used in PCR

37
Q

True or False:

cDNA can be used in PCR

A

True

38
Q

True or False:

cDNA cannot be used in PCR

A

False

cDNA can be used in PCR

39
Q

What is it called in PCR when the two strands of template DNA are separated?

A

Denaturation

40
Q

What names are given to the two strands of the template DNA?

A

Positive strand and negative strand

41
Q

Give the names of the four d deoxynucleotides

A

dATP
dTTP
dGTP
dCTP

42
Q

What occurs during the extension stage of PCR?

A

Taq polymerase uses the dNTPs to extend the newly synthesised chain of DNA, using the primers as an anchor.

43
Q

In each cycle of a PCR, what happens to the number of DNA molecules?

A

It doubles

44
Q

How many cycles would a normal PCR have?

A

30-40 cycles

45
Q

Fill in the gaps: PCR Process

  1. Genomic or cDNA which is being used as a _______ is put in a ____
  2. Heat to _____°C to _____________________ (denaturation)
  3. Add ________ which are complimentary to the positive and negative strands of the DNA
  4. Cool to around ___°C to allow them to anneal to complimentary sequence
  5. Add _______________ (dNTPs), dATP, dCTP, dTTP, dGTP
  6. Temperature raised to ___°C to allow Taq polymerase uses primers as an ______ to extend _____ __________ chain of DNA using the ______ (extension)
  7. Repeat cycle – each cycle _______ the DNA. A normal PCR will include around ______ cycles
A
  1. Genomic or cDNA which is being used as a template is put in a tube
  2. Heat to 90-95°C to separate the two strands of template DNA (denaturation)
  3. Add primers which are complimentary to the positive and negative strands of the DNA
  4. Cool to around 55°C to allow primers to anneal to complimentary sequence
  5. Add d deoxynucleotides (dNTPs), dATP, dCTP, dTTP, dGTP
  6. Temperature raised to 70°C to allow Taq polymerase uses primers as anchor to extend newly synthesized chain of DNA using the dNTPs (extension)
  7. Repeat cycle – each cycle doubles the DNA. A normal PCR will include around 30-40 cycles
46
Q

How do we know if the PCR was successful?

A

Use agarose gel electrophoresis on the products of the PCR to separate them by size

47
Q

Give two advantages of a PCR

A

Very sensitive, could start with one target molecule and amplify to billions of copies

Very specific, because the primers must match the target gene

48
Q

Why is PCR so specific?

A

The primers must match the target gene

49
Q

Why is PCR so sensitive?

A

Could start with only one target molecule and amplify to billions of copies

50
Q

How could PCR be used to identify whether meat is beef or horse meat?

A

Use primers designed for sequences which are specific from one species.

51
Q

How could PCR be used to identify disease alleles?

A

Design primers to only anneal to the normal allele (or the disease allele)

Could potentially detect alterations caused by mutations / insertions / deletions

52
Q

How could PCR be used in forensics?

A

If there is very little DNA available to start with, PCR can amplify what is available, increasing the amount that can be used for different tests

53
Q

How could PCR be used for gene expression analysis?

A

… … …

54
Q

Who is the Southern blotting technique named after?

A

Ed Southern, who developed the technique

55
Q

Why is the Southern blotting technique useful?

A

It allows the identification of specific DNA fragments in a complex mixture (such as an entire genome of DNA)

56
Q

Which technique combines electrophoresis and nucleic acid hybridisation to detect the gene or fragment of interest in a complex mix of thousands of DNA fragments (such as an entire genome of DNA)?

A

Southern blotting method

57
Q

What is nucleic acid hybridisation?

A

DNA probes labelled with fluorescent dye or a radioactive isotope such as 32P, which are complementary to the gene of interest, are annealed to the sample DNA after it is also denatured so we can detect the presence of sample DNA.

58
Q

Fill in the gaps: Southern blotting 1/2
_______ _______ DNA and cut with restriction enzymes to generate thousands of different fragments.
Separate fragments by ____ using _______________.
________ the DNA using _____________ to enable it to _____________ from the Agarose gel into a membrane (usually _______ _______).
We use a ______ ______ to promote the capillary transfer to the membrane, called a _____.
Use ___ to fix the DNA to the membrane once it is inside.

A

Isolate genomic DNA and cut with restriction enzymes to generate thousands of different fragments.
Separate fragments by size using an agarose gel electrophoresis.
Denature the DNA using an alkaline solution to enable it to transfer by capillary from the Agarose gel into a membrane (usually nitrous cellulose).
We use a heavy weight to promote the capillary transfer to the membrane, called a blot.
Use UV to fix the DNA to the membrane once it is inside.

59
Q

Fill in the gaps: Southern blotting 2/2
The membrane will produce a blot containing the same pattern of bands from the original gel.
_________ gene of interest with a radioactive probe, which is a small piece of ____________, synthetic, __________ labelled DNA (___), complementary to the sequence of interest.
__________ _____ can be used to detect the signal from the labelled probe and produce a _____ _______ which can be __________ to the fragments of the gene of interest from known cells with known alleles.

A

The membrane will produce a blot containing the same pattern of bands from the original gel.
Hybridize gene of interest with a radioactive probe, which is a small piece of single-stranded, synthetic, phosphorus labelled DNA (P32), complementary to the sequence of interest.
Photographic film can be used to detect the signal from the labelled probe and produce a band pattern which can be compared to the fragments of the gene of interest from known cells with known alleles.

60
Q

What is DNA sequencing used for?

A

Determination of the order of bases in a strand of DNA, usually in a 5’ to 3’ direction

61
Q

What does the Sanger dideoxynucleotide method of DNA sequencing require?

(5 things)

A
  1. Single stranded DNA template
  2. Primer
  3. dNTPs
  4. ddNTPs (low concentration) (dideoxynucleotides) (modified dNTPs)
  5. DNA polymerase
62
Q

What principle is the sanger dideoxynucleotide method of DNA sequencing is based on?

A

The principle of primer extension

63
Q

The nucleoside triphosphates have something the dideoxynucleotides do not. What is it and why is it important?

A

Have the 3’ hydroxyl group in the sugar

This acts as the attachment site for the phosphorus from the next nucleotide. Allows sugar phosphate backbone to be generated, so the chain in the newly synthesised strand can grow

64
Q

The modified ddNTPs do not contain something the nucleoside triphosphates (dNTPs) do.
Why is this important?

A

Don’t have the 3’ hydroxyl group in the sugar

Formation of next sugar phosphate bond is not possible, terminating the elongation capacity of the polymerase

65
Q

What does dNTP mean?

A

Deoxynucleotide (nucleoside triphosphate)

66
Q

What does ddNTP mean?

A

Dideoxynucleotide

67
Q

Fill in the gaps: DNA sequencing 1/2
Add the DNA template strand, the complementary primer, the dNTPs, the ________ tagged ddNTPs and the DNA polymerase together.
Primer finds complementary sequence and _________ with template DNA.
DNA polymerase starts _________ the new chain incorporating ______.
By chance a ______ will be incorporated somewhere in place of a _____, __________ the chain.
Iteration of this process many times results in the polymerase eventually _________________.
Each fragment will be labelled differently depending on the ___________ on the ______.

A

Add the DNA template strand, the complementary primer, the dNTPs, the fluorescently tagged ddNTPs and the DNA polymerase together.
Primer finds complementary sequence and base pairs (or anneals) with template DNA.
DNA polymerase starts elongating the new chain incorporating dNTPs.
By chance a ddNTP will be incorporated somewhere in place of a dNTP, terminating the chain.
Iteration of this process many times results in the polymerase eventually terminating fragments at every position.
Each fragment will be labelled differently depending on the fluorescent tag on the ddNTP.

68
Q

Fill in the gaps: DNA sequencing 2/2
Fragments are organised from _______ to _______ and injected into a _______ that will also organise them _______ to ________.
A laser and detector are used to record the _______________ through the ________ as each fragment passes through the laser one at a time.
Since each ______ has a different ___________ associated with it we can get a read of the sequence by analysing the order of the different ________________ associated to each ddNTP.

A

Fragments are organised from shortest to longest and injected into a capillary that will also organise them shortest to longest.
A laser and detector are used to record the fluorescence colour through the capillary as each fragment passes through the laser one at a time.
Since each ddNTP has a different fluorescence associated with it we can get a read of the sequence by analysing the order of the different fluorescent peaks associated to each ddNTP.

69
Q

True or False:

During DNA sequencing, more dNTPs should be added to the mix than ddNTPs

A

True

70
Q

True or False:

During DNA sequencing, more ddNTPs should be added to the mix than dNTPs

A

False

During DNA sequencing, more dNTPs should be added to the mix than ddNTPs

71
Q

How are ddNTPs tagged in DNA sequencing?

A

Each type of ddNTP is fluorescently tagged with a different colour to allow for differentiation between them

72
Q

What is cycle sequencing? (DNA sequencing)

A

Normal DNA sequencing but DNA template is amplified by PCR first. Increases copies of fragments so during laser step more fluorescent fragments are present, increasing intensity of reading

BIOL114 Biotechnology (10 decks)

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