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Biology 311 > Lectures II-i > Flashcards

Lectures II-i Flashcards

1
Q

what is the purpose of polymerase chain reaction?

A

amplify selected regions of DNA in vitro

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2
Q

what does the initial solution of PCR contain?

A

DNA template, primers, nucleotides, heat-tolerant DNA polymerase

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3
Q

describe one cycle of PCR

A

1) heat to denature DNA to make single-strand DNA (95)
2) cool to let primers hybridize/anneal to complementary sequences (50-65)
3) polymerase replicates the single strand DNA, extending from primer (72)

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4
Q

dideoxy sequency/Sanger sequency

A

using a specially modified nucleotide (ddNTP) that blocks DNA synthesis because it does not have 3’ hydroxyl group to determine sequence of DNA

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5
Q

gel electrophoresis

A

fragments are ordered by increasing length, smaller fragments move faster

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6
Q

point mutation

A

alteration of a single base pair of DNA or of a small number of adjacent base pairs

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7
Q

what are the two types of point mutations?

A
  1. base substitution (one base pair is replaced by another)

2. insertion/deletion mutations

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8
Q

what are two types of base substitutions?

A

transition (replacement of bases of the same category) and transversion (replacement of a base of other category)

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9
Q

spontaneous mutations

A

naturally occurring, arises in all cells

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10
Q

induced mutations

A

arise through the action of certain agents (mutagens) which increases the rate of mutation

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11
Q

what are possible mechanisms for spontaneous mutations?

A

error in DNA replication (base substitution, insertion/deletion, frameshift mutations), spontaneous lesion (depuration & deamination), or oxidatively damaged bases

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12
Q

mutagenesis

A

production of mutations through exposure to mutagens

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13
Q

what are mechanisms of mutagenesis?

A

incorporation of base analogs that alters pairing properties, specific mispairing, intercalating agents (planar molecules that mimic base pairs and cause insertion/deletion), base damage (no specific base pairing is available, blocks replication)

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14
Q

locus

A

a location in the genome (single or multiple nucleotides)

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15
Q

single nucleotide polymorphisms (SNP’s)

A

difference in nucleotide present at a single nucleotide site (most prevalent type of polymorphism)

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16
Q

genetic variability

A

chemical changes/differences within our genome

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17
Q

what causes genetic variability?

A

change in # of copies of genes/chromosomes, creation of new alleles, change in expression pattern of genes

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18
Q

what are changes to DNA passed on to the next generation?

A

because of the semiconservative nature of DNA (where one strand acts as the template for the synthesis of the other)

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19
Q

forward primer directionality vs. reverse primer directionality

A

forward: extended left to right
reverse: extended right to left

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20
Q

agarose gel electrophoresis separates DNA fragments on the basis of:

A

size

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21
Q

in gel electrophoresis, distance migrated in DNA is …

A

inversely proportional to fragment size (increased size=increased friction so less movement)

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22
Q

higher agarose concentration results in:

A

smaller pores

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23
Q

band thickness varies with:

A

concentration

24
Q

use DNA ladder to construct:

A

a standard curve for size vs. distance travelled to determine size of DNA fragments in experiment

25
Q

+ control checks:

A

if PCR worked and locus was successfully identified, target sequence is obtained

26
Q
  • control checks:
A

if primer worked (controls for genetic variability, some people have different sequences at the locus)

27
Q

H2O control checks:

A

to make sure there is no contamination

28
Q

to set up DNA sequence experiment, add:

A

DNA template, taq polymerase, nucleotides (dNTP), a SINGLE DNA primer, a different ddNTP to four different tubes

29
Q

when results of DNA sequencing experiment is run on an agarose gel, the results obtained are:

A

the sequence for the complimentary daughter strand (read top down to get 5’-3’ parent strand sequence)

30
Q

gene

A

a coding locus (producing either a protein or functional RNA)

31
Q

anonymous locus

A

DNA locus without any apparent function

32
Q

changes in the DNA sequences occurs in what type of locus?

A

any type

33
Q

the locus is POLYMORPHIC if:

A

two or more different DNA sequences exist at a locus

34
Q

DNA polymorphisms

A

changes in the DNA sequence

35
Q

single nucleotide polymorphisms (SNPs)

A

most common, replacing one nucleotide with another

36
Q

simple sequence length polymorphisms (SSLPs)

A

sequence of nucleotides that is present in a number of repeats that can vary in the number of repeats from one person to the next

37
Q

large -scale chromosomal changes

A

(duplication, inversion, translocation, deletion), source of copy number variants CNVs

38
Q

what causes SNPs

A
  1. DNA polymerase adds an incorrect base, results in a mismatched base pair
  2. base tautomerization, results in mismatch after second round of replication-after paired with base and results in future mismatch in additional rounds of replication (unstable tautomer reverts back to stable form)
  3. spontaneous and/or induced lesions
39
Q

g* looks like

A

A so G* pairs with T

40
Q

how are lesions introduced?

A

by replacing a base in DNA, by altering a base so that it specifically mispairs with another base, by damaging a base so that it can no longer pair with any base

41
Q

what are three main types of SSLPs?

A
  1. microsatellites (2-3 nucleotides)
  2. short tandem repeats STR (4-6 nucleotides)
  3. variable number tandem repeats VNTR (dozens of nucleotides)
    the number of units of repeats for the sequences above varies
42
Q

what causes SSLPs

A

slippage at various loci within a repeating unit at the replication fork (occurs at repeats because the ends are still able to bond together so DNA molecule is stable)

43
Q

if slippage occurs in the daughter strand:

A

insertion

44
Q

if slippage occurs during the template strand:

A

deletion

45
Q

effects of slippage are seen in:

A

the second round of replication

46
Q

CNV (copy number variants) include:

A

repeats and duplications that increase copy number and deletions that reduce copy number (of individual genes, sets of genes, or entire genes)

47
Q

do CNV’s affect the genetic message?

A

not usually

48
Q

what does RFLP stand for?

A

restriction fragment length polymorphism, a technique that uses a restriction enzyme to detect SNP’s

49
Q

what does RFLP detect?

A

SNP’s

50
Q

procedure of RFLP

A
  1. obtain amplified locus you want to study 2. incubate for certain amount of time at specific temperature 3. run products on agarose gel
51
Q

what do you put in a tube for RFLP

A

amplified DNA of interest, appropriate restriction enzyme, buffer to create optimal environment for enzyme

52
Q

RFLP with Southern blot

A

restriction fragments are run out on a gel, products are denatured and transferred to filter, a PROBE is used to detect restriction fragments of interest

53
Q

what is a microarray

A

basically a southern blot on a microchip

54
Q

how techniques can be used to detect SSLP’s?

A

agarose gel electrophoresis (to detect differences in size/length), sequencing, RFLP (also detects differences in size of fragments), southern blotting (basically electrophoresis with a probe), southern blotting with RFLP

55
Q

restriction enzymes recognize:

A

specific DNA sequences called restriction sites

56
Q

polymorphisms can create or destroy:

A

restriction sites

57
Q

when incubated with restriction enzymes, different alleles can give:

A

different patterns of bands on a gel

Biology 311 (6 decks)

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