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Biochemistry Block 4 > Recombinant DNA Technology > Flashcards

Recombinant DNA Technology Flashcards

1
Q

Biotechnology tools are used routinely for what 2 type of diagnoses ?

A

Biochemical and genetic

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2
Q

Restriction endonucleases:

  1. What do they do
  2. How long are the recognized sequences generally
  3. What else is characteristic of the sequences
A
  1. Cleave DNA at very specific DNA sequences
  2. Usually only 4-8 base pairs long
  3. They are palindromes
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3
Q
  1. Restriction enzymes cleave DNA and leave what 2 ends?

2. __ or __ remain attached to cleavage products after cleavage, this is important for?

A
  1. Sticky ends and blunt ends

2. 3’ OH group or 5’ phosphate; ligation reactions

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4
Q

How are restriction enzymes generally named?

Define restriction site

A

For the organism they were derived from

The DNA sequence that can be cleaved by a restriction enzyme

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5
Q

Restriction enzyme with other biochemical tools are used to do what 3 things

A

Cut, paste, and analyze DNA

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6
Q
  1. What is recombinant DNA
  2. Easier with __ ends, why?
  3. What does DNA ligase do in this case
A
  1. Fragments of DNA pasted together to make hybrid molecules
  2. Sticky; makes it easier to paste the DNA to another piece of DNA
  3. Creates the phosphodiester bonds
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7
Q
  1. DNA cloning involves
  2. What then happens to the vector
  3. This causes the DNA to be __ and __
  4. What is this process called?
A
  1. Inserting restriction fragment into a cloning vector
  2. Vector can then be replicated in host cells (usually bacteria)
  3. Cloned and amplified
  4. Recombinant DNA amplification
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8
Q
  1. What are vectors?
  2. Must be capable of __ replication in the cell
  3. Must have at least __ for foreign DNA insertion
  4. Must carry what?
A
  1. Molecules of DNA that can accept fragments of foreign DNA (by cutting the restriction site)
  2. Autonomous (make it recognizable by the bacteria)
  3. One restriction site
  4. One gene for selection (usually antibiotic resistance)
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9
Q

What are the most common vectors

4 other vectors

Each __ has one vector

A

Prokaryotic plasmids (bacteria)

Phages, yeast plasmids, yeast artificial chromosomes (YACs) and mammalian viruses

Bacteria cell (so there are a lot of vectors)

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10
Q

What are the 2 types of DNA libraries?

A

Genomic DNA libraries and cDNA libraries

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11
Q

Genomic DNA libraries:

  1. What 2 things happen to the genome in order to be used to transform bacteria
  2. Each transformed bacteria containing a __ may contain different segment of the genome
  3. What does the collection contain?
A
  1. The entire genome is chopped up with restriction enzymes and cloned into vectors
  2. Plasmid
  3. All sequences in the genome
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12
Q

cDNA (complementary DNA) libraries:

  1. How is cDNA generated?
  2. MRNA is reverse transcribed by ___; second strand is synthesized by ___
  3. How is cDNA used to transform bacteria
  4. Ideally, a cDNA library contains sequences representing what?
  5. How does DNA polymerase synthesize the second strand
A
  1. Using isolated mRNA from a cell/ tissue type of interest (by reverse transcription*)
  2. Reverse trancriptase; DNA polymerase
  3. It is ligated into a vector
  4. All mRNAs present in the cell/tissue type at the time mRNA was collected
  5. By using the complementary strand as the template
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13
Q

Recap: What is reverse transcription

A

Making DNA from mRNA

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14
Q

cDNA libraries:

  1. What kind of cells would you collect cDNA from usually
  2. Libraries only contain cDNA sequences that represent __
A
  1. Ones that are being affected by a certain condition (to see what genes were being expressed in diseased cells)
  2. The mRNA sequences (no introns, promotors, etc.)
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15
Q
  1. DNA from cDNA library can be cloned into __ for __

2. What 3 things are used to transform expression bacteria strains (bacterial expression vector)

A
  1. An expression vector for production of proteins

2. Promotor, shine delgarno sequence, and cDNA

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16
Q

DNA sequencing:

  1. Used to determine
  2. DNA is melted to generate
  3. 4 major components of the reaction used to conduct sequencing
  4. Split sample into 4 tubes that all contain a small amount of a specific?
A
  1. Exact sequence of a cloned or PCR amplified stretch of DNA
  2. Single-stranded template
  3. DNA, dNTPs, DNA primer, and polymerase
  4. Dideoxyribonucleotide
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17
Q

Based on size, what runs thru the gel fastest?

A

Smaller=faster; bigger=slower

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18
Q

Probes:

  1. Used to identify
  2. What type of molecule is a probe
  3. Labeled using __
  4. Has the ability to do what?
A
  1. DNA fragments
  2. ssDNA
  3. Radioactivity
  4. Hybridize with other pieces of ssDNA (complementary base pairing = hybridization)
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19
Q

Hybridization:

  1. DNA of interest is made into __
  2. Why is target DNA then immobilized on a solid support?
  3. When exposed to probe, what will happen if complementary sequence is present?
A
  1. SsDNA
  2. So it cannot reanneal with its original complementary strand
  3. Probe will bind the immobilized ssDNA (identified via autoradiography)
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20
Q

Type of probes: smaller probes

  1. Chemically synthesized __; how many bases
  2. Very specific - they identify __
A
  1. Oligonucleotides; 2-30 bases

2. Only detect the nucleotide sequences that it will base pair with

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21
Q

Type of probes: larger probes

  1. How are they made - give 2 examples
  2. Less specific - can identify __ or __
A
  1. Via molecular biology techniques - reverse transcription, PCR, etc.
  2. Similar genes in different organisms or same gene in different individuals that may not be exactly the same sequence
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22
Q

Southern blotting:

  1. Analysis of
  2. Digested DNA is subjected to __
  3. What then happens to DNA
  4. Reason for this?
A
  1. DNA
  2. Gel electrophoresis
  3. It is denatured and blotted (immobilized onto a membrane)
  4. Allows the DNA to be probed to see if it hybridizes
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23
Q

Northern blotting:

  1. Analysis of
  2. Do not need to make it __, why? But it must be __
  3. Only detects __ sequences
  4. Can be used for __ or __ specific studies
A
  1. RNA
  2. Single stranded (because RNA is already single stranded), but must be complementary to mRNA
  3. Expressed
  4. Tissue or cell
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24
Q

Western blotting:

  1. Analysis of
  2. Prove is an __ specific to protein of interest - usually attached to an __, why?
  3. Qualitative or quantitative
A
  1. Protein
  2. Antibody; enzyme to identify positive reaction
  3. Quantitative
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25
Q

Restriction fragment length polymorphisms (RFLPs):

  1. When you do a restriction digest when there is a polymorphism, there is a difference in __
  2. Genetic differences are due to __ or __
  3. RFLPs are usually not caused by
  4. Most of the genome is various types of _
A
  1. The length of the fragments that are generated
  2. Mutations or polymorphisms
  3. Disease mutations
  4. Intervening sequences
26
Q

RFLPS:

  1. Why are genetic variations in intervening sequences generally not harmful
  2. Why are intervening sequences hyper-variable
  3. Define polymorphisms
  4. Term “mutation” is reserved for
A
  1. They have no consequence on the expression of genes (proteins)
  2. No selection pressure for genetic changes
  3. Genetic variations in non-coding regions with no disease
  4. Genetic changes that cause disease
27
Q

RFLP is present if a genetic change in a polymorphic region:

  1. Creates or deletes a ___; meaning what is no longer present?
  2. Has more or less of a ___
A
  1. Restriction site; palindrome

2. Repeated sequence

28
Q

DNA variations resulting in RFLPs: Single nucleotide polymorphisms (SNPs):

  1. What is an SNP
  2. Accounts for 90% of
  3. May create or abolish a ___
  4. Harmless or dangerous ?
A
  1. Single nucleotide changes
  2. Genetic variation
  3. Restriction site
  4. Usually harmless, occasionally created by a disease causing a mutation
29
Q

DNA variations resulting in RFLPs: Variable number of tandem repeats (VNTRs)

  1. Genome contains many regions where a sequence is
  2. Does it vary greatly from person to person
  3. How will a VNTR produce an RFLP
A
  1. Repeated in tandem many times
  2. Yes (unique for individual)
  3. If DNA is cleaved on either side of a VNTR, an RFLP is produced
30
Q

Summary-SNPs:

  1. Creation or abolishment of __
  2. Different size RFLPs based on different?
  3. Used to ?
A
  1. A restriction site
  2. Restriction cutting
  3. Mark genes (alleles), disease markers
31
Q

Summary-VNTRs:

  1. More or less of a
  2. Different size RFLPs based on the different
  3. Used as
A
  1. Tandem repeat
  2. Number of repeats
  3. Molecular fingerprints (identifying individuals)
32
Q

Compare and contrast the following:

  1. DNA cloning amplifies __ by
  2. PCR is?
  3. Which is quicker?
A
  1. Fragments by inserting them into a vector and the host replicating the vector
  2. Amplification done completely in a tube; uses heating/cooling to amplify
  3. PCR
33
Q

Primers for PCR:

  1. Primers act as a primer for which enzyme
  2. What do you need to know about the sequence you wish to amplify?
  3. Which primers are synthesized; how many nucleotides?
A
  1. DNA polymerase
  2. Need to know the sequence of 2 small flanking regions on the sequence you wish to identify
  3. The ones that are complementary to the flanking regions; ~20
34
Q

PCR steps:

  1. __ DNA into separate strands
  2. Anneal primers to __ of ssDNA
  3. __ with DNA polymerase
  4. 2 new double stranded DNAs can be __ and __ by steps 1-3
A
  1. Denature
  2. “Flanking regions”
  3. Extend primers
  4. Denatured and copied
35
Q

Why do you denature the DNA for PCR?

A

Produce ssDNA so the primers can bind to it

36
Q

DNA polymerase uses __ to extend the primer and build ___

A

dNTPs; complementary copy of the DNA strand

37
Q

***So in simplistic terms, what are steps 2-4 of PCR (one word each)

A

Denaturing, annealing, and extension

38
Q

What type of DNA polymerases are used for PCR? Why?

A

Taq DNA polymerases; they are heat stable

39
Q

Advantages of PCR:

  1. Sensitivity meaning?
  2. Speed - how long does it take compared to cloning?
  3. Name 4 uses
  4. Other advantage
A
  1. Only need trace amounts of DNA (single cell)
  2. Only takes a few hours - cloning takes weeks
  3. Mutation detection, detection of latent viruses (HIV), forensics, prenatal genetic diagnosis
  4. Can use the DNA for other things
40
Q
  1. Northern blot looks at __

2. Identify this based on ?

A
  1. Expression of a specific gene

2. The probe you’re using

41
Q

Analysis of gene expression: microarrays

  1. __ levels
  2. Contain __ on glass slides
  3. Used to compare __ changes in different cell types
  4. Synthesize and label __ from 2 different cell types with different fluorescent probes
A
  1. mRNA
  2. Thousands of immobilized sequences (probes)
  3. “Global” gene expression changes
  4. cDNA

~so mRNA is being converted to cDNA (reverse transcription) to detect a green (normal) color and red (diseased) color

42
Q

Gene expression profile for a particular component:

  1. Green spot indicates?
  2. Red spot
  3. Yellow spot
  4. Black spot
A
  1. Normal cell produces more of its message
  2. Cancer cell produces more of its message
  3. Both cells produce the same amount of their message
  4. Neither cell produces this message
43
Q

Big picture for what you are looking at for microarrays

A

Looking at multiple genes that are upregulated or downregulated relative to a specific scenario

44
Q

Proteomics - what is it

Similar to?

A

Proteomics- looks at different proteins produced in particular cell/tissue types
Similar to genomics

45
Q

ELISAs:

  1. __ is bound to the well of a microtiter plate
  2. Probed with __
  3. Detect by adding ___ to form a colored reaction
  4. Used as a screening tool because it is
A
  1. Antigen (protein)
  2. Antibody linked to an enzyme
  3. Substrate for enzyme
  4. Very sensitive

~so antigen is the target, probe identifies the target - so ELISA detects the presence of an antigen

46
Q

Verify ELISA with what type of blot?

A

Western

47
Q

Western blot:

  1. Analysis of
  2. Protein samples are separated by gel based on __
  3. Membrane is probed with __ to identify bands
  4. More specific than ELISA but
A
  1. Proteins
  2. Size
  3. An enzyme linked antibody
  4. More time/labor intensive and less sensitive
48
Q

What could give you a false positive on ELISA

A

Antibody has the ability to interact with something else in the background of the wells

49
Q

Sickle cell anemia:

  1. __ a restriction site for what restriction enzyme
  2. What causes the RFLP in this case; used for what
  3. In most diseases, RFLP used for diagnosis is linked to the disease, but not ___
A
  1. Eliminates
  2. The disease causing mutation; used for diagnosis
  3. The actual disease causing mutation
50
Q

What has a larger band on southern blot: normal or sickle cell anemia

A

Sickle cell anemia

51
Q

What kind of probes are used to diagnose sickle cell anemia

A

ASO probes (allele specific oligonucleotide)

52
Q

ASO probes:

  1. Specific for __
  2. Can recognize?
  3. Can determine if someone does or does not __
A
  1. An allele
  2. Single nucleotide differences
  3. Have a mutation that could result in disease
53
Q

Cystic fibrosis:

  1. Caused by
  2. Results in defective
  3. Due to ___ of CFTR
  4. Most common __ mutation in caucasians
A
  1. Genetic mutation
  2. Cl ion channeling
  3. Improper folding and assembly
  4. Lethal genetic mutation (delta f 508 mutation)
54
Q

Cystic fibrosis:

  1. Elevated __ levels in sweat
  2. In lungs, lack of Cl secretion leads to
  3. Predisposed to
  4. Also causes __ insufficiency
  5. How is it diagnosed
A
  1. Chloride
  2. Dehydration of the mucus
  3. Recurrent lung infections
  4. Pancreatic enzyme insufficiency
  5. Using ASO probes
55
Q

PCR for Cystic fibrosis

  1. PCR using primers specific to ___
  2. How does PCR affect the product of a homozygous affected person
  3. What will PCR produce for a heterozygote
A
  1. The region of deletion
  2. Gives a shorter than normal product (missing 3 bases)
  3. Both a normal and mutant allele of the CTFR gene product means PCR will produce 2 products
56
Q

PKU:

  1. Will have high levels of __ and low levels of __
  2. Mutations occur in any of the __ and __ are larger than normal
A
  1. High of phenylalanine and low of tyrosine

2. 13 exons; introns

57
Q

In order to screen for PKU you must have what 2 things

A

DNA from several family members/affected individual

An identifiable marker linked with the particular mutation in this family (ie an RFLP)

58
Q

Marker for PKU is not actually the disease causing mutation, marker would normally be? Define this term

A

A polymorphism - nucleotide change that results in an added or lost restriction site

59
Q

Myotonic dystrophy:

  1. What type of disease
  2. Because it is this kind of disease, it will produce what, where?
A
  1. Trinucleotide repeat expansion disease (in 3’ non-coding region of a protein kinase gene)
  2. RFLPs (when digested with a restriction enzyme) on both sides of the expansion
60
Q
  1. How is HIV diagnosed
  2. Initial test used? Second test?
  3. What can occur with ELISA
  4. What can you use to test for HIV immediately
  5. What is quantitative PCR used to monitor?
A
  1. Immunoassay - so antibody recognition test basically
  2. ELISA then western blot
  3. False positives
  4. PCR
  5. Used to monitor viral load in HIV positive patients
61
Q

__ is sensitive; __ is specific

A

ELISA is sensitive; western blot is specific

62
Q

Paternity testing:

  1. Use __ as molecular fingerprints
  2. Usually done using __ opposed to __
A
  1. Several different VNTRs

2. PCR opposed to southern blotting

Biochemistry Block 4 (9 decks)

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