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Biochemistry > DNA Technology > Flashcards

DNA Technology Flashcards

1
Q

DNA technology permits

A

Manipulation and analysis of DNA, synthesis and mutation of genes, clone and express genes to produce proteins, locate gene mutations, detect carriers of genetic disease

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2
Q

Enzymes used in DNA technology

A

Restriction endonucleases, DNA ligase, DNA polymerase, Reverse transcriptase

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3
Q

Restriction endonucleases

A

Restriction enzymes, cut double-stranded DNA into smaller fragments, cut at specific sequence- restriction site, isolated from bacteria

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4
Q

Restriction site

A

Specific sequence of bases cleaved by a restriction enzyme, most commonly 4-6 base pairs long, many are palindromic (both strands have same sequence when read 5’ to 3’)

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5
Q

Sticky ends

A

Restriction enzyme cuts at palindromic restriction site, leaves staggered ends of the DNA

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6
Q

Blunt ends

A

Restriction enzymes cuts evenly through both strands of DNA

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7
Q

DNA ligases

A

Join together fragments of DNA, in vivo they repair single-stranded breaks, in vitro then join fragments of double-stranded DNA

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8
Q

Ligation

A

Joining together DNA molecules, more efficient between sticky ends, can add linkers to blunt ends to make them sticky

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9
Q

DNA polymerases

A

Makes copies of DNA, synthesize a single new strand complementary to an existing single-stranded DNA template, most require a double-stranded primer

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10
Q

Reverse transcriptase

A

Copies RNA onto DNA, synthesizes a DNA strand complementary to an RNA template, used in production of complementary DNA (cDNA), required for production of recombinant eukaryotic proteins in prokaryotes

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11
Q

Analyzing the result of a DNA reaction

A

Reaction products are separated by gel electrophoresis, DNA molecules move according to their size, smaller the molecule, the faster it moves, bands are visualized

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12
Q

Separation of products by gel electrophoresis

A

DNA loaded into a well cut out of the gel, DNA separates into bands of different-sized fragments

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13
Q

Estimation of product size

A

Size markers- mix of DNA fragments of known, different lengths

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14
Q

Hybridization techniques

A

Techniques involving annealing of two single strands of complementary base sequence, involve use of probe to identify or select specific DNA fragments

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15
Q

Probe

A

Short single-stranded nucleotide of known sequence, usually labelled to permit identification

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16
Q

Southern blotting

A

Identification of DNA fragments through their ability to hybridize with a complementary probe

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17
Q

Northern blotting

A

Identification of RNA fragments

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18
Q

Western blotting

A

Identification of proteins through their ability to bind to specific antibodies

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19
Q

Applications of southern blotting

A

Identifying genes, diagnosis of genetic diseases, carrier detection, DNA fingerprinting

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20
Q

DNA profiling (fingerprinting)

A

Original method used probes that annealed to DNA regions comprising repeated blocks of bases, number of repeats varies from one individual to another

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21
Q

Minisatellite DNA

A

Comprises variable number tandem repeats (VNTRs)

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22
Q

Microsatellite DNA

A

Compromises short tandem repeat polymorphism (STRPs)

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23
Q

DNA sequencing

A

Determination of the sequence of bases in a DNA molecule, Maxam-Gilbert chemical method, chain termination or dideoxy method, automated sequencing using fluorescence

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24
Q

DNA sequencing protocol

A

4 sequencing reactions are set up, one for each base, each reaction is set up to synthesize a nucleotide chain complementary to the chain under analysis, all reaction components are added to each reaction plus a small amount of ddNTP

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25
Q

Automated DNA sequencing

A

Based on Sanger method, employs fluorescent labeling, different fluorolabel attached to each ddNTP, all 4 reactions performed in one tube and electrophoresis performed in one lane of the gel

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26
Q

Polymerase chain reaction (PCR)

A

In vitro method for producing large amounts of DNA from a target sequence, requires only minute amount of sample to be amplified, can produce a million copies in 2-3 hours

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27
Q

PCR reaction components

A

Target DNA template, deoxynucleotide mixture (dATP, dTTP, dCTP, dTTP), heat stable DNA polymerase, oligonucleotide primers complementary to ends of region to be amplified

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28
Q

Steps of PCR

A

Heat separates strands, add synthetic oligonucleotide primers, cool, add thermostable DNA polymerase to catalyze DNA synthesis, repeat, DNA synthesis catalyzed by thermostable DNA polymerase, target sequence amplified 10^6 fold after 25 cycles

29
Q

Advantages of PCR

A

Size and condition of sample, speed

30
Q

Disadvantages of PCR

A

Some sequence knowledge required, size limit for target DNA, contamination

31
Q

Uses of PCR

A

Accelerating and facilitating genetic engineering, diagnosing genetic diseases, identifying carriers of genetic diseases, detecting presence of infectious organisms, DNA profiling

32
Q

Vectors

A

Vehicles that carry DNA fragments into cells- plasmid, bacteriophage, cosmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), vectors for gene therapy (viruses)

33
Q

Transduction

A

Phage mediated gene transfer into a prokaryotic cell

34
Q

Transformation

A

Non-phage mediated gene transfer into prokaryotic cell

35
Q

Transfection

A

Gene transfer into a eukaryotic cell

36
Q

Formation of recombinant DNA molecule

A

Add gene to vector with DNA ligase, forming a recombinant DNA molecule

37
Q

Clone

A

Group of organisms, cells, or molecules all originating from a single individual

38
Q

Clone a gene

A

Isolate the DNA that encodes the gene in a pure, readily reproducible form, can be used for amplifying DNA or production of the encoded protein from cloned DNA

39
Q

DNA libraries

A

Large collection of recombinant DNA clones, in which the DNA has been inserted into a vector

40
Q

Genomic library

A

A collection of DNA fragments from an organism’s entire genome cloned into bacterial colonies, contains all genes for an organism

41
Q

cDNA library

A

Collection of cDNA fragments produced from all the mRNA present in a particular tissue type, contains only cDNAs corresponding to proteins expressed in the cell type from which the library was made

42
Q

Screening a genomic library

A

Selecting a colony that contains a specific individual gene, hybridization with an oligonucleotide probe

43
Q

Screening a cDNA library

A

Polymerase chain reaction, hybridization with an oligonucleotide probe, immunological screening

44
Q

Medical applications of DNA technology

A

Diagnosis of genetic diseases, carrier detection, detection of infectious organisms, production of therapeutic proteins, production of DNA vaccines, gene therapy

45
Q

DNA techniques in diagnosis

A

Most involve preliminary amplification by PCR- restriction fragment length polymorphism (RFLP) analysis, direct PCR analysis, allele-specific oligonucleotide (ASO) hybridization, hybridization to microchip array

46
Q

Detection of mutation by direct PCR analysis

A

PCR amplification of mutated region

47
Q

Allele-specific PCR amplification (ARMS test)

A

PCR equivalent of ASO hybridization, achieves the same purpose in one PCR step, 2 sets of primers: 1st primer the same for both reactions, 2nd primer in 2 forms, normal and mutant, only exact complementary target sequence is amplified

48
Q

PCR target size limitations

A

Routine use, not >4000 bases, disorders due to triplet nucleotide expansions, full mutation too large to amplify routinely

49
Q

DNA microarrays

A

Used to detect mutations and to examine gene expression, normal and mutated ASOs robotically placed on glass slide, hybridized with fluorescently labeled DNA from patient, pattern analyzed by computer

50
Q

Therapeutic proteins produced by recombinant DNA technology

A

Insulin, factor VIII, factor IX, human growth hormone, tissue plasminogen activator, interferon, erythropoietin, adenosine deaminase

51
Q

Production of vaccines by recombinant DNA technology

A

Protein antigens can be produced completely free of the infectious agent, hepatitis B vaccine was first produced recombinant DNA vaccine

52
Q

Gene therapy

A

To cure or prevent a disease by modifying an individual’s genes or their expression, somatic cell therapy or germline therapy

53
Q

Somatic cell therapy- gene replacement therapy

A

Replacing a mutated gene that causes disease with a healthy gene

54
Q

Somatic cell therapy- gene therapy for non-inherited diseases

A

Introducing a new gene to help fight a disease

55
Q

Somatic cell therapy- gene-blocking therapy

A

Inactivating a mutated gene that is functioning improperly

56
Q

Retrovirus vectors

A

Advantages- enters cells efficiently, integrates stably into host genome, targets only dividing cells
Disadvantages- limited insert size, integrates into host genome, targets only dividing cells

57
Q

Adenovirus vectors

A

Advantages- enters cells efficiently, high expression of insert, does not integrate into host genome
Disadvantages- can elicit serious immune response, does not integrate into host genome

58
Q

Adeno-associated virus vectors

A

Advantages- enters cells efficiently, elicits little or no immune response, integrates into host genome at specific site
Disadvantages- limited insert size, difficult to produce

59
Q

Herpes simplex virus vectors

A

Advantages- can carry up to 20kb, prolonged activity, infects nerve cells efficiently
Disadvantages- can cause immune response

60
Q

Liposome vectors

A

Advantages- can accept large inserts, no immune response

Disadvantages- inefficient cell entry, no integration into host genome, can be toxic

61
Q

Naked DNA vectors

A

Advantages- can accept large inserts, no immune response

Disadvantages- very inefficient cell entry, no integration into host genome

62
Q

Viral vs non-viral vectors

A

Non-viral vectors are better suited for ex vivo gene therapy than in vivo gene therapy

63
Q

Ex vivo gene therapy

A

Patient’s cells are extracted, manipulated outside the body, and re-introduced, used for SCID, familial hypercholesterolemia, Gaucher disease, malignant melanoma, leukemia

64
Q

In vivo gene therapy

A

Used for cystic fibrosis, various cancers (brain tumors, ovarian cancer, neck cancer), hemophilia B, Duchenne muscular dystrophy

65
Q

Success with human gene therapy

A

SCID, malignant melanoma, Leber’s congenital amaurosis, hemophilia B, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), Parkinson disease, cystic fibrosis

66
Q

Gene therapy for lipoprotein lipase deficiency

A

Glybera approved as treatment, delivers lipoprotein lipase in an adeno-associated virus (AAV) vector, one time intramuscular injection

67
Q

Transgenic animals and plants

A

Organisms with foreign DNA introduced in the genome, can increase growth rate, disease resistance, produce nutritional supplements

68
Q

Compounds produced in pharm animals

A

Human hemoglobin, human tissue plasminogen activator (TPA)

Biochemistry (18 decks)

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