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B4 Biochem > Recombinant DNA Technology and Biotechnology > Flashcards

Recombinant DNA Technology and Biotechnology Flashcards

1
Q

Biotechnology has made enormous contributions to ____________

A

Medicine

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2
Q

Important achievements leading to modern molecular biology

A

Restriction endonucleases

Cloning of DNA

Creation of Synthetic probes

PCR

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3
Q

Enzymes that cleave very specific DNA sequences

A

Restriction enzymes

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4
Q

What is the length and style of restriction endonucleases?

A

Generally short, about 4-8 BP

Usually palindromes

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5
Q

What does it mean when it says “restriction enzymes are usually palindromes?”

A

DNA palindromes read the same 5’ to 3’ on both strands.

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6
Q

Is this example a palindrome?

5’ TCTAGA 3’
3’ AGATCT 5’

A

Yes

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7
Q

Is this example a palindrome?

5’ TCTTCT 3’
3’ AGAAGA 5’

A

Nope

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8
Q

What does it mean when a restriction enzyme cleaves a “sticky” end?

A

There are unpaired nucleotides hanging off each end.

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9
Q

What does it mean when it says restriction enzymes cleave to make “blunt” ends?

A

The bases are paired with each other after cleavage.

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10
Q

Restriction enzymes are typically named for…

A

The organism they were derived from.

Eco RI -> E Coli

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11
Q

DNA sequences that can be cleaved by a restriction enzyme

A

Restriction site

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12
Q

In theory, which restriction enzyme would cut more frequently?

A restriction enzyme that recognizes a 4 base recognition sequence

OR

A restriction enzyme that recognizes a 6 base recognition sequence

A

The 4 base recognition sequence. The shorter the sequence, the more often it is cleaved.

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13
Q

What molecule is formed when fragments of DNA are “pasted” together?

With what time of cleavage is this easiest?

A

Recombinant DNA

Sticky ends

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14
Q

Enzyme that creates the phosphodiester bonds in recombinant DNA

A

DNA ligase

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15
Q

The insertion of a restriction fragment into a cloning vector

A

DNA cloning

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16
Q

Significance of DNA cloning

A

The vector can now be replicated in host cells (usually bacteria, or yeast)

DNA is now cloned and amplified.

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17
Q

Molecules of DNA that can accept fragments of foreign DNA

A

Vector

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18
Q

What are 3 things that a vector MUST do?

A

Must be capable of autonomous replication in the cell

Must have at least one restriction site for foreign DNA insertion

Must carry at least one gene for selection

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19
Q

What gene do vectors USUALLY carry for selection?

A

Antibiotic resistance

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20
Q

What are the most common vectors?

A

Prokaryotic plasmids

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21
Q

Other vectors

A

Phages

Yeast plasmids

Yeast artificial chromosomes

Mammalian viruses

(Retroviruses)

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22
Q

2 types of DNA libraries

Which are very different from each other

A

Genomic DNA libraries

cDNA libraries

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23
Q

How Genomic DNA Libraries are made:

A
  1. The entire genome is chopped up with restriction enzymes
  2. The cleaved parts of the genome are cloned into vectors
  3. These vectors then transform bacteria
  4. The transformed bacteria contains thousands of different segments of the genome
  5. The collection in the bacteria is therefore a Library, containing all sequences of the genome (including coding regions as well as introns and other intervening sequences, promoters, etc)
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24
Q

What is cDNA?

A

Complementary DNA

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25
Q

cDNA is generated using….

A

Isolated mRNA from a particular cell of tissue

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26
Q

Process of creating cDNA

A
  1. mRNA is reverse transcribed and the second strand is synthesized to make it double stranded.
  2. cDNA is lighted into a vector, used to transform bacteria and 1000s of clones are collected
  3. cDNA library is created, containing sequences representing all mRNAs present in the specific cell or tissue type
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27
Q

What enzyme reverse transcribes mRNA?

A

Reverse transcriptase

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28
Q

What enzyme crates the second strand of cDNA?

A

DNA polymerase

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29
Q

Purpose of cDNA libraries

A

Allows one to see what genes were being used in a particular cell or tissue type

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30
Q

cDNA libraries ONLY contain ____ sequences.pr

A

mRNA (no introns, promoters, etc)

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31
Q

DNA from a cDNA library can be cloned into an expression vector for:

A

Production of proteins

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32
Q

Promoter, Shine-Dalgarno sequence, and cDNA are used to transform expression bacteria strains

A

Bacterial expression vectors

Huh?

33
Q

Mammalian vector type hosts

A

Yeast, ex: HBV

34
Q

Technique used to determine the exact sequence of a cloned or PCR amplified stretch of DNA

A

DNA sequencing

35
Q

Steps of DNA sequencing

A
  1. DNA is melted to generate SS template
  2. Samples containing DNA, dNTPs, DNA Primer, and polymerases are split into 4 tubes
  3. Each tube contains a small amount of specific dideoxyribonucleotides (ddCTP, ddATP, ddGTP, ddTTP)
  4. conduct a PCR sequencing reaction
  5. Apply the samples to an agarose gel for electrophoresis
36
Q

An ssDNA molecule, used to identify DNA fragments, that is labeled using radioactivity that can be hybridized

A

Probes

37
Q

Hybridization using probes

A
  1. Target DNA is made SS (using heat/chemicals)
  2. Target is immobilized on a solid support (nitrocellulose membrane) so it cannot reanneal with its original complementary strand
  3. The ssDNA-coated membrane is exposed to the probe
  4. If complementary strand is present, probe will bind to the immobilized ssDNA and can be identified via autoradiography
38
Q

Probes that are chemically synthesized oligonucleotides (2-30 bases), very specific

A

Smaller probes

39
Q

Probes that are made via one of several molecular biology techniques, and much less specific

A

Larger probes

40
Q

How are smaller probes made?

A

Same way synthetic primers are made

41
Q

How are larger probes made?

A

Reverse transcription, PCR, etc.

42
Q

3 different blotting techniques

A

Southern blotting

Northern blotting

Western blotting

43
Q

Blotting for analyzing DNA

A

Southern blotting

  1. Dna is isolated and subject to restriction digestion
  2. Digested DNA is ten subjected to gel electrophoresis
  3. DNA is denatured, blotted (immobilized onto a membrane)
  4. Blot membrane is probed
44
Q

Blotting used to analyze RNA

A

Northern blotting

Do NOT need to make SS

Probe MUST be complementary to mRNA

ONLY detects expressed sequences

Can be used for tissue or cell studies

Is quantitative

45
Q

Blotting used to analyze Proteins

A

Western blots

Probe is an antibody specific to the protein of interest

Usually attached to an enzyme to identify positive reaction on. Blot

Quantitative

46
Q

What is an RFLP?

A

Restriction Fragment Length Polymorphisms

47
Q

Genetic variations in non-coding regions with no disease

A

Polymorphisms

48
Q

Genetic changes that cause disease

A

Mutation

49
Q

What 2 genetic changes in a polymorphic region will cause an RFLP to be present?

A
  1. Restriction site is created or deleted

2. There is more or less of a repeated sequence

50
Q

2 DNA variations resulting in RFLPs

A
  1. Single nucleotide polymorphisms (SNPs)

2. Variable Number of Tandem Repeats (VNTRs)

51
Q

Single nucleotide changes that account fo 90% of genetic variation

May create or abolish a restriction site

A

SNPs

52
Q

When a human genome contains many regions where a sequence is repeated in tandem many times.

Varies greatly from person to person and is unique for an individual

If DNA is cleaved on either side of a VNTR, an RFLP is produced

A

VNTRs

53
Q

RFLPs based on SNPs

A

Creation of abolishment of a restriction site

Different size RFLPs based on different restriction cutting

Used to mark genes/alleles, as disease markers

54
Q

RFLPs based on VNTRs

A

More or less of a tandem repeat

Different size RFLPs based on the different number of repeats

Used as a molecular fingerprints (identifying individuals)

55
Q

What revolutionized molecular biology?

A

PCR, bitch.

Freakin Kary Mullins won a nobel prize, yo.

56
Q

What is the main difference between DNA cloning and PCR?

A

DNA cloning is done by using a host to replicate a vector

PCR is amplified completely in a tube

57
Q

To make a primer for PCR, first we must know..

A

The sequence of two small flanking regions of the sequence we wish to amplify

58
Q

Steps of PCR

A
  1. Denature DNA into separate strands
  2. Anneal primers to “flanking regions” of ssDNA
  3. Extend primers with DNA polymerase
  4. The two new dsDNA molecules can be denatured by repeating steps 1-3 (AKA CHAIN RXN)
59
Q

What is the flanking region?

A

An area of ssDNA where the primer attached to (in PCR)

60
Q

First step of PCR is to…

A

Denature the target DNA, produce ssDNA.

Via heat

61
Q

The second step of PCR is to…

A

Anneal the primers.

The ssDNA has primers attached to its flanking regions

62
Q

The 3rd step of PCR is to..

A

Extend the chain via DNA polymerase.

Uses dNTPs to extend the primer and build a complementary copy of the DNA strand

63
Q

Steps 1-3 (which are what?) are repeated how many times?

A

Denature, anneal, extend

Around 20-30 times.

64
Q

What specific enzymes are used for PCR?

A

Heat stable Taq DNA polymerases

Due to the fact the temp changes through each step. For ex:

  • denaturization = 95 C
  • annealing = 55 C
  • extension = 72 C
65
Q

What are some advantages of PCR?

A

Sensitivity (only trace amounts of DNA needed, like a single cell)

Speed (only takes a few hours, compared to cloning taking weeks)

DNA that is produced can be used for numerous other tests like gel electrophoresis, southern blotting, etc

Practical uses, such as mutation detection, virus detection (HIV), forensics, prenatal genetic diagnosis

66
Q

Analysis of gene expression in Northern Blots

Is this a qualitative or quantitative test?

What are you looking for?

Can you look at numerous genes at once?

A

Checking mRNA levels..

Both

Checking the expression of a specific gene, depending on the probe used.

Only one gene at a time.

67
Q

If you see a blotting technique that represents checking RNA…

A

It must be a northern blot

68
Q

Technique used that contains thousands of immobilized sequences (probes) on glass slides

A

Microarrays

69
Q

To check mRNA levels, what method is used for gene expression?

A

Microarrays.

mRNA undergoes reverse transcriptase to create cDNA then the cDNA is label with a fluorescent dye and placed in the microarrays for analysis

Used to compare “global” gene expression changed in different cell types

Analyzed on a computer

70
Q

Similar to genomics but looks at all the different proteins produced in particular cell or tissue types

A

Proteomics

71
Q

Enzyme-linked immunosorbent assays

Very sensitive, not very specific.

A

ELISAs

72
Q

In an ELISA, an _________ is bound to the wall of a ___________ plate.

A

Antigen

Microliter

73
Q

In ELISA, how is the antigen “probed”?

A

With an antibody linked to an enzyme

74
Q

How does an ELISA work?

A

The antigen is probed with an antibody linked to an enzyme, and detects by adding substrate for enzyme to form a colored reaction

75
Q

Western Blot

A

Analyzes proteins of gene expression

Protein samples are separated by gel electrophoresis based on size

Proteins are blotted onto membrane

Membrane is probed with an enzyme-linked antibody to identify bands

MORE specific than ELISA, but more time and labor, less sensitive

76
Q

Which methods of genetic testing are quantitative?

A

Northern blot, western blot, microarray, ELISA, proteomics

77
Q

What is so special about Sickle cell anemia in genetic testing?

A

Sickle cell mutuation is in the B-globin gene, and it eliminates the restriction site for MstII (rest. Enzyme)

Creates an RFLP that can be used for diagnosis, as compared to most RFLPs that are linked to the disease.

78
Q

Different ways to test for sickle cell….

A

Southern blot - probe beta-globin gene, check for larger band

PCR- amplify mutated region, digest products with MstII, check for larger band

ASO probes- use allele specific oligonucleotides to hybridize with mutated region (fast and reliable)

B4 Biochem (2 decks)

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