Lectures II-i

Question 1

what is the purpose of polymerase chain reaction?

Answer 1

amplify selected regions of DNA in vitro

Question 2

what does the initial solution of PCR contain?

Answer 2

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

Question 3

describe one cycle of PCR

Answer 3

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)

Question 4

dideoxy sequency/Sanger sequency

Answer 4

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

Question 5

gel electrophoresis

Answer 5

fragments are ordered by increasing length, smaller fragments move faster

Question 6

point mutation

Answer 6

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

Question 7

what are the two types of point mutations?

Answer 7

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

2. insertion/deletion mutations

Question 8

what are two types of base substitutions?

Answer 8

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

Question 9

spontaneous mutations

Answer 9

naturally occurring, arises in all cells

Question 10

induced mutations

Answer 10

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

Question 11

what are possible mechanisms for spontaneous mutations?

Answer 11

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

Question 12

mutagenesis

Answer 12

production of mutations through exposure to mutagens

Question 13

what are mechanisms of mutagenesis?

Answer 13

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)

Question 14

locus

Answer 14

a location in the genome (single or multiple nucleotides)

Question 15

single nucleotide polymorphisms (SNP’s)

Answer 15

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

Question 16

genetic variability

Answer 16

chemical changes/differences within our genome

Question 17

what causes genetic variability?

Answer 17

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

Question 18

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

Answer 18

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

Question 19

forward primer directionality vs. reverse primer directionality

Answer 19

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

Question 20

agarose gel electrophoresis separates DNA fragments on the basis of:

Answer 20

size

Question 21

in gel electrophoresis, distance migrated in DNA is …

Answer 21

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

Question 22

higher agarose concentration results in:

Answer 22

smaller pores

Question 23

band thickness varies with:

Answer 23

concentration

Question 24

use DNA ladder to construct:

Answer 24

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

Question 25

+ control checks:

Answer 25

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

Question 26

- control checks:

Answer 26

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

Question 27

H2O control checks:

Answer 27

to make sure there is no contamination

Question 28

to set up DNA sequence experiment, add:

Answer 28

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

Question 29

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

Answer 29

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

Question 30

gene

Answer 30

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

Question 31

anonymous locus

Answer 31

DNA locus without any apparent function

Question 32

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

Answer 32

any type

Question 33

the locus is POLYMORPHIC if:

Answer 33

two or more different DNA sequences exist at a locus

Question 34

DNA polymorphisms

Answer 34

changes in the DNA sequence

Question 35

single nucleotide polymorphisms (SNPs)

Answer 35

most common, replacing one nucleotide with another

Question 36

simple sequence length polymorphisms (SSLPs)

Answer 36

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

Question 37

large -scale chromosomal changes

Answer 37

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

Question 38

what causes SNPs

Answer 38

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

Question 39

g* looks like

Answer 39

A so G* pairs with T

Question 40

how are lesions introduced?

Answer 40

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

Question 41

what are three main types of SSLPs?

Answer 41

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

Question 42

what causes SSLPs

Answer 42

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)

Question 43

if slippage occurs in the daughter strand:

Answer 43

insertion

Question 44

if slippage occurs during the template strand:

Answer 44

deletion

Question 45

effects of slippage are seen in:

Answer 45

the second round of replication

Question 46

CNV (copy number variants) include:

Answer 46

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

Question 47

do CNV's affect the genetic message?

Answer 47

not usually

Question 48

what does RFLP stand for?

Answer 48

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

Question 49

what does RFLP detect?

Answer 49

SNP's

Question 50

procedure of RFLP

Answer 50

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

Question 51

what do you put in a tube for RFLP

Answer 51

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

Question 52

RFLP with Southern blot

Answer 52

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

Question 53

what is a microarray

Answer 53

basically a southern blot on a microchip

Question 54

how techniques can be used to detect SSLP's?

Answer 54

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

Question 55

restriction enzymes recognize:

Answer 55

specific DNA sequences called restriction sites

Question 56

polymorphisms can create or destroy:

Answer 56

restriction sites

Question 57

when incubated with restriction enzymes, different alleles can give:

Answer 57

different patterns of bands on a gel