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MFM > Molecular and Biochemical Techniques > Flashcards

Molecular and Biochemical Techniques Flashcards

1
Q

what does agarose gel electrophoresis do??

A

allows for separation of DNA fragments based on SIZE, the DNA migrates through the gel via electric current from negative (top) to positive (bottom) and moves around pores in the gel

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2
Q

what is PCR?

A

polymerase chain reaction, amplifies a specific sequence of DNA exponentially, helpful when you don’t have a lot of product and need to analyze it

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3
Q

what diagnostic testing is PCR used for?

A

presence of infectious agents like virus, bacteria based on the nucleic acids present in the agent, helpful because the amplification power of PCR allows a very low level of infection to be detected early in the infection process (not a window period)

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4
Q

what is a window period?

A

time period between the initial infection and the production of antibodies by the infected individual’s body

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5
Q

how many copies of a gene will there be after 35 cycles?

A

68 billion copies, takes less than 2 hours

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6
Q

what is the the downside of using ELISA or western blot diagnostic tests for infections?

A

they require presence of antibodies to the infective agent for a positive result, but the body has a window period where the production of Ab has not started yet, but the person is still infected, so can’t detect infection early

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7
Q

what are the 5 components of PCR?

A
  1. DNA template of the sequence to be amplified
  2. DNA polymerase - Taq polymerase because it is heat stable
  3. pool of dNTPs (dATP, dTTP, dCTP, dGTP)
  4. 2 primers to start DNA replication process, oriented in opposing directions
  5. buffer for optimal salt concentration and pH, and Mg2+ cofactor (for DNA polymerase activity)
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8
Q

what are restriction enzymes?

A

enzyme that cleaves DNA phosphodiester bond at specific nucleotide sequences, leaving sticky ends, can be used to identify variations in the sequences of genes, like RFLP (restriction fragment length polymorphisms)

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9
Q

what do restriction enzymes cleave?

A

usually recognize palindrome sequences

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10
Q

what are sticky ends and why is that important?

A

products are single stranded at the end (not blunt), and are complimentary so they can be ligated back together with other sticky ends

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11
Q

what is restriction fragment length polymorphisms

A

a diagnostic test for presence of certain diseases like sickle cell anemia, if a mutation occurs in a recognition site for a restriction enzyme, the DNA from the mutated person cannot be cleaved, so the fragments produced will be a different length than wild type, so the fragments can be assessed using southern blotting technique. if the restriction enzyme cleaves different length fragments there is a mutation in that area

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12
Q

explain sickle cell anemia in relation to RFLP

A

single nucleotide substitution in beta globin gene eliminates a MstII restriction enzyme recognition site, so the DNA will be cleaved in different locations, thus showing up on a southern blot

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13
Q

what is VNTR?

A

variable number of tandem repeats, or regions of genomic DNA that are repeated in tandem a variable number of times

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14
Q

how is DNA fingerprinting done?

A

restriction enzyme sites flanking VNTR’s can be cut to produce DNA fragments of varying lengths, and each individual has a different pattern of DNA fragment!!

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15
Q

what is recombinant DNA?

A

when two fragments of DNA with sticky ends from restriction enzyme cleavage are ligated to form a hybrid of 2 original DNA molecules

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16
Q

what are recombinant DNA used for?

A

experimental proteins and DNA, for therapeutic proteins and incorporation of DNA of interest

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17
Q

steps of DNA cloning

A
  1. specific piece of DNA is amplified, needs a carrier DNA or plasmid/circular DNA
  2. both DNA interest and vector are cut with the same restriction enzyme
  3. DNA of interest is joined to vector creating hybrid molecule (chimeric plasmid)
  4. chimeric plasmid introduced into bacterial cells
  5. bacterial cells replicate the chimeric plasmid - exponential amplification
  6. plasmid purified from bacteria OR bacteria induced to make protein from the DNA that was cloned into the vector
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18
Q

benefit of tagging with fluorescent proteins?

A

allows for visualization of cellular structures in living cells, because the protein of interest is connected to the fluorescent protein

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19
Q

how to integrate green fluorescent protein into alpha-tubulin?

A
  1. start with alpha tubulin complementary DNA and a plasmid containing GFP gene and a promoter sequence
  2. cut both with same restriction enzyme (EcoRI)
  3. join sticky ends of alpha tubulin cDNA to sticky ends of the plasmid
  4. creates cloned cDNA consisting of a promoter and the genes (cDNA) of alpha tubulin and GFP fused together in frame
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20
Q

how can therapeutic recombinant proteins be made in large quantities?

A

using bacteria to amplify the gene of the protein of interest

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21
Q

process of synthesizing therapeutic proteins

A
  1. gene encoding protein of interest placed in plasmid that is amplified in bacteria
  2. bacteria grown in large quantities, millions of copies of plasmid
  3. bacteria induced to produce protein from gene of interest in plasmids
  4. large quantities of encoded recombinant protein made
  5. recombinant protein is then purified from bacteria and modified if needed (isolated) (need to modify outside bacterial cell since it cannot itself)
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22
Q

what therapeutic proteins can be made by recombination and “cloning” and amplifying?

A

insulin - one of the first therapeutic recombinant proteins made
also made factor VIII, tissue plasminogen activator (TPA), and EPO

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23
Q

what are nucleic acid probes?

A

short fragments of ssDNA or ssRNA that recognize a specific sequence in target DNA, these probes are labeled with either fluorescent tag or by incorporation of a radioactive nucleotide

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24
Q

what must occur with target DNA before it can recognize a nucleic acid probe?

A

must be treated with heat or alkali conditions to produce single strands, so sequence on probe can bind

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25
Q

what is fluorescence in situ hybridization (FISH)

A

single stranded DNA probes that are conjugated to fluorescent molecule are made that recognize sequence of interest in the chromosome (gene or mutation), so you can see the location and number of genes/mutation on a chromosome under a fluorescent microscope

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26
Q

how is FISH used in diagnostics?

A

can be used to detect presence or absence of specific sequences of DNA in chromosomes, or deletions like activating mutations in proto-oncogenes and loss of tumor suppressor genes, AND chromosomal translocations and amplifications associated with specific diseases!

27
Q

what are examples of things that can be seen in FISH

A

ERBB2 gene amplification in sporadic breast cancer (HER2, Neu)
BCR-ABL translocations/philadelphia chromosomes in CML

28
Q

what is southern blot

A

method of detecting specific sequences of DNA

29
Q

what is northern blot

A

method of detecting specific sequences of RNA

30
Q

what is western blot

A

method of detecting specific proteins

31
Q

steps of southern or northern blots

A
  1. nucleic acid samples separated on an agarose gel by electrophoresis
  2. after the separation, the DNA/RNA is transferred from gel to nitrocellulose membrane
  3. a probe is made that has sequence complimentary to the sequence of interest in the sample, is labeled with fluorescence or radioactive nucleotides
  4. labeled probe is incubated with membrane containing transferred DNA samples
  5. membrane is exposed to autoradiography film and DNA/RNA fragments that are bound by the labeled probe are visible via labeled bands
32
Q

how can southern blotting be used for diagnostics?

A

can ID genetic diseases with known mutations, like presence of sickle cell

33
Q

what is sickle cell caused by?

A

point mutation in beta globin gene, eliminates the recognition site for the restriction enzyme MstII, which leaves only 2 MstII sites (where normal has 3), so if a gene is mutated, there will only be 1 large band

34
Q

what does a normal beta globin gene look like on a southern plot?

A

the normal gene has 3 MstII sites, and therefore would produce 2 small bands (probe hybridizes to region between MstII gives three possible bands on southern blot)

35
Q

what is a dideoxyribose and what occurs if it is incorporated into a chain? (ddNTP)

A

a deoxyribose but with no hydroxy groups (OH), so it cannot be incorporated into a DNA chain because it cannot bind to another nucleotide, CAUSES CHAIN TERMINATION

36
Q

what is the overall charge of DNA?

A

negative, because the phosphate group in backbone contains a free O- at physiological pH

37
Q

what is the purpose of sanger sequencing?

A

can have particular size of DNA fragments and where the chains terminate (represents the place where the ddnucleotide was added) and this can be plotted by agarose gel electrophoresis and DNA sequence can be read from bottom to top (5-3), this can be compared with the known wild type sequence and see what has changed or mutated

38
Q

what is the sanger method

A

amplification of target DNA sequence in the presence of specific modified nucleotides (ddNTPs), causes chain termination because the ddNTP doesnt have 3-OH needed to incorporate the next nucleotide, therefore the fragments are of varying lengths (random replacement), and the products are separated by agarose gel electrophoresis and the DNA sequence can be read from bottom to top

39
Q

what is polyacrylamide gel electrophoresis (PAGE)

A

allows for separation of proteins based on size when they are loaded on a polyacrylamide gel (that is porous when polymerized), and the migration of the protein is mediated by electrical current where the top is negative the bottom is positive (the smaller proteins are at the bottom)

40
Q

what is necessary before PAGE

A

proteins must be converted from their secondary structure to make them uniform/standard, and the plate must be coated with SDS negative charge to make the charges all negative (neutral) - this ensures the proteins are sorted by size only

41
Q

steps of western blots

A

1/2. protein samples are loaded onto polyacrylamide gel and separated by size (electric current), small on bottom, large on top

  1. gel removed from electrophoresis apparatus and proteins from gel are transferred to a membrane, making a direct copy (proteins are in same location)
  2. membrane is incubated with primary antibodies that are specific for protein of interest, then incubated with secondary antibodies that are conjugated to an enzyme
  3. colorimetric or chemiluminescent enzyme substrate added
  4. signal is seen where target protein is on gel, seeing size and amount of the proteins
42
Q

how can western blots be used for diagnostics?

A

HIV antibody test, lyme disease, and other infectious diseases

43
Q

process of HIV antibody test

A

blood is taken from the human, Ab are separated from blood and used as probe. known virus proteins are separated on polyacrimide gel and if the patient’s Ab recognize these viral proteins, they are HIV positive

44
Q

what is the downside of using western blots for diagnostics?

A

there is a risk of a false negative during the seroconversion window period where Ab are not being developed despite the human being infected - early detection is not possible

45
Q

what is ELISA?

A

enzyme linked immunosorbent assay, a very sensitive method for detecting the presence of an antibody or protein

46
Q

steps of ELISA

A
  1. antigen of interest bound to the bottom of a plate
  2. patient’s sample (blood containing primary Ab) added to plate
  3. secondary Ab conjugated to an enzyme is added
  4. colorimetric or chemiluminescent substrate added
  5. signal color indicates antigen of interest is present in patient
47
Q

what can ELISA be used to detect?

A

autoimmune diseases, test donated blood for HIV, rapid antibody tests for HIV (home microELISA technology also) (western blot must be used to confirm diagnosis)

48
Q

False positives for HIV using ELISA

A

person can have Ab in blood that non-specifically bind to HIV antigen for some reason

49
Q

false negatives for HIV using ELISA

A

testing is too early in the infection process

50
Q

what is a southwestern blot

A

used to ID protein-DNA interactions (ex. can see transcription factor binding to gene promoted)

51
Q

process of southwestern blot

A
  1. sample run on SDS-PAGE gel to separate proteins based on size
  2. separated proteins transferred to membrane (like western blot)
  3. double stranded DNA probes of specific sequences are then added to blot (like southern blot)
  4. if probes bind to protein on the blot, that means that the protein can bind the specific DNA sequence found in that probe
52
Q

what is microarray

A

if you want to compare 2 conditions’ gene expression (when exposed to 2 different conditions or between health and diseased cell), ssDNA molecules representing all expressed genes in a cell are fixed to glass slide/silicon film and used as reference

53
Q

procedure of microarray

A
  1. isolate mRNA from 2 samples
  2. use reverse transcriptase to make DNA copy of mRNA (cRNA) that is labeled with dye (different for each of the strands)
  3. labeled cDNAs hybridized to reference DNA chip
  4. if spot is red, that specific gene as expressed at higher level in red-dyed sample
  5. if spot is green, that specific gene was expressed at higher level in green-dyed sample
  6. if yellow, specific gene expressed at same level in both samples
54
Q

what is proteomics?

A

compare differences in expression of proteins between two samples

55
Q

procedure of proteomics?

A
  1. proteins from 2 different samples isolated and each sample labeled with different fluorescent dye
  2. proteins separated by 2D gel electrophoresis (1 dimension = charge, 2 dimension = size)
  3. computer protein aligns spots and determines which proteins are upregulated or downregulated based on the intensity of the spot
56
Q

what is gene therapy?

A

adding another normal copy of gene to genome when there is a defective copy of said gene by incorporating normal into a viral particle and making the viral particle infect the patient’s cells, and once inside the gene is processed into a functional protein via transcription/translation

57
Q

what viral vectors are usually utilized in gene therapy?

A

retroviruses, adenoviruses

58
Q

what are retroviruses

A

RNA viruses that use reverse transcriptase to make dsDNA copy of their RNA genome, and can stably integrate into a cell’s nuclear DNA, making PERMANENT THERAPY using small genes

59
Q

problem with retroviruses as vectors

A

integration site is random, so can cause problems if integrated into area that disrupts a gene or causes overexpression of a gene, and can only infect actively dividing cells

60
Q

what are adenoviruses

A

DNA virus that do NOT integrate into cell’s genome, can carry larger genes and can infect both dividing and nondividing cells

61
Q

problem with adenoviruses

A

do not integrate, so no permanent effect, so have to repeat tx

62
Q

compare retrovirus and adenovirus as gene therapy vector

A

retrovirus is permanent that integrates into the cell’s nuclear DNA, but it can only carry small genes and is integrated randomly and only infects actively dividing cells.
adenovirus not permanent that does not integrate into cell’s genome, can carry larger genes, and can infect dividing and non-dividing cells

63
Q

implication of transgenic technology

A

possible to produce organisms in which specific gene has been replaced by mutated version or in which function of a specific gene is completely removed, as in knock out mice

MFM (39 decks)

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