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QUIZ 6 > Molecular Techniques > Flashcards

Molecular Techniques Flashcards

1
Q

restriction enzymes

A

cleave palindromic sequences with a double stranded cut; made by bacteria

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2
Q

unexpected pattern of digestion

A

indicates polymorphism - deletion or substitution, insertion, methylation change

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3
Q

agarose gel electrophoresis

A

separates DNA based on size; longer DNA will move shorter distances

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4
Q

separated DNA segments used for _

A

cloning, generating a probe, purifying a desired segment for DNA sequence determination

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5
Q

capillary electrophoresis

A

electrophoretic DNA separation without a gel

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6
Q

DNA cloning

A

ability to generate desired DNA fragments of DNA

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7
Q

DNA cloning steps

A

restriction enzymes cleave restriction site to produce sticky sites –> foreign DNA with gene of interest contains matching sticky ends so it will hybridize –> recombinant vector carrying gene of interest is placed in presence of antibiotics –> only recombinant vectors will grow and divide because of antibiotic resistance gene

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8
Q

plasmid components

A

OriC, restriction site, and antibiotic resistance gene

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9
Q

What is useful for cloning large segments of DNA?

A

artificial chromosomes

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10
Q

What is useful for cloning shorter sequences of DNA?

A

PCR and recombination methods

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11
Q

DNA library

A

cloned segments from entire DNA or RNA (cDNA) complement of target of interest carried in a vector

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12
Q

libraries can be made from _

A

genomic DNA, cDNA from total mRNA, or subset of either

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13
Q

DNA hybridization (heteroduplex)

A

ssDNA is annealed to complementary single stranded sequence

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14
Q

Southern Blot

A

restriction enzymes digest genomic DNA and fragments are separated in agarose gel –> sequences are transferred to Southern blot for hybridization

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15
Q

Southern blot is used for _

A

specific detection of specific sequences (ex. sickle cell)

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16
Q

Western blot

A

proteins

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17
Q

Northern blot

A

RNA

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18
Q

selective hybridization

A

can be used with small segments of DNA to detect single mismatches

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19
Q

How does selective hybridization work?

A

if the DNA segment of interest will not anneal to the known complementary DNA, it is because there is a mismatch which indicates disease

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20
Q

PCR steps

A

denature, anneal to primer, extending stage

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21
Q

PCR requirements

A

thermostable polymerase, single stranded primer, dNTPs and needed enzymes

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22
Q

PCR can also be _

A

quantitative; determine how much was in original sample

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23
Q

real-time quantitative PCR

A

the more you start with, the less cycles you will need to get to a detection point (exponential phase)

24
Q

limitations of PCR

A

must be able to define endpoints with two primers, any error will be amplified, GC sequences difficult to amplify, polymerases eventually fail

25
Q

dideoxy nucleotides

A

both 2’ and 3’ C’s lack hydroxyl groups, terminating the chain when they bind

26
Q

Sanger dideoxy sequencing

A

four tubes with low concentrations of radio-labeled ddNTPs are mixed with regular dNTPs and DNA polymerases –> DNA will elongate until reaches a ddNTP –> eventually results in multiple lengths with a known end which can tell you the sequence of the DNA

27
Q

limitations of Sanger sequencing

A

sequence obtained is of newly synthesized strand, rather than template, so errors possible, polymerases don’t last forever, not large enough for genome-scale analysis

28
Q

exome

A

complete exon complement

29
Q

detection of large DNA segments

A

chromosome banding karyotype (>100kb)

30
Q

detection of specific large DNA segments

A

southern blot hybridization or FISH (>10kb)

31
Q

balanced translocation

A

all information is there but in a different spot

32
Q

differential detection of normal vs. altered sequences or sequence copy number

A

selective or allele-specific hybridization, CGH

33
Q

amplification of defined or less defined set(s) of sequences

A

CGH, PCR, DNA sequencing

34
Q

unbalanced translocation

A

missing material

35
Q

isochromosomes are examples of _

A

a balanced translocation

36
Q

paracentric inversion

A

rearrangement on one side of centromere (within p or q arm)

37
Q

pericentric inversion

A

rearrangement around the centromere (p to q or vice versa)

38
Q

FISH and karyotyping require _

A

metaphase cells because the chromosomes are condensed

39
Q

When can FISH be done with interphase cells?

A

if looking for duplication or loss

40
Q

comparative genome hybridization

A

used to detect copy number changes only

41
Q

karyotyping uses _

A

detecting suspected or unknown aneuploidy and large structural rearrangements (deletions, inversions, translocations)

42
Q

karyotype disadvantages

A

difficulty in automation, must use metaphase cells (takes a while to induce)

43
Q

chromosome paint

A

FISH that covers the whole chromosome in certain color (probe sequences must be unique so color is not repeated on different chromosome)

44
Q

RFLP

A

used to identify suspected changes (gene level) –> based on Southern blot hybridization

45
Q

comparative genome hybridization (CGH)

A

can be used when you have no idea what’s wrong but will only detect copy number variations

46
Q

CGH method

A

sample DNA and reference DNA are hybridized –> normal genes will hybridize completely and form mixed color

47
Q

too much sample DNA in CGH

A

indicated a duplication

48
Q

too much reference DNA in CGH

A

indicates deletion

49
Q

SNPs

A

single nucleotide polymorphisms

50
Q

methods to detect SNPs

A

exome sequencing, whole genome sequencing, targeted enrichment for sequencing, gene-specific microarray

51
Q

micro-array based mutation scanning

A

will show the nucleotide in a normal sequence and then will show the nucleotide in diseases sequence

52
Q

targeted gene sequencing

A

sample DNA is cut into fragments and allowed to hybridize with reference DNA –> targeted sequences are eluded and amplified –> determine sequence

53
Q

advantages of targeted gene sequencing

A

efficient for scanning many segments or long genome regions, detects all smaller variant types, and less expensive than more global sequencing techniques

54
Q

whole exome sequencing

A

all annotated exons are targeted

55
Q

methods to detect changes in cytosine methylation

A

sodium bisulfite treatment followed by PCR and DNA sequencing of products

56
Q

sodium bisulfite

A

will cause unmethylated cytosines to deaminate into uracils –> will eventually have T’s in PCR analysis

QUIZ 6 (11 decks)

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