Molecular Techniques

Question 1

restriction enzymes

Answer 1

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

Question 2

unexpected pattern of digestion

Answer 2

indicates polymorphism - deletion or substitution, insertion, methylation change

Question 3

agarose gel electrophoresis

Answer 3

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

Question 4

separated DNA segments used for _

Answer 4

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

Question 5

capillary electrophoresis

Answer 5

electrophoretic DNA separation without a gel

Question 6

DNA cloning

Answer 6

ability to generate desired DNA fragments of DNA

Question 7

DNA cloning steps

Answer 7

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

Question 8

plasmid components

Answer 8

OriC, restriction site, and antibiotic resistance gene

Question 9

What is useful for cloning large segments of DNA?

Answer 9

artificial chromosomes

Question 10

What is useful for cloning shorter sequences of DNA?

Answer 10

PCR and recombination methods

Question 11

DNA library

Answer 11

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

Question 12

libraries can be made from _

Answer 12

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

Question 13

DNA hybridization (heteroduplex)

Answer 13

ssDNA is annealed to complementary single stranded sequence

Question 14

Southern Blot

Answer 14

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

Question 15

Southern blot is used for _

Answer 15

specific detection of specific sequences (ex. sickle cell)

Question 16

Western blot

Answer 16

proteins

Question 17

Northern blot

Answer 17

RNA

Question 18

selective hybridization

Answer 18

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

Question 19

How does selective hybridization work?

Answer 19

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

Question 20

PCR steps

Answer 20

denature, anneal to primer, extending stage

Question 21

PCR requirements

Answer 21

thermostable polymerase, single stranded primer, dNTPs and needed enzymes

Question 22

PCR can also be _

Answer 22

quantitative; determine how much was in original sample

Question 23

real-time quantitative PCR

Answer 23

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

Question 24

limitations of PCR

Answer 24

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

Question 25

dideoxy nucleotides

Answer 25

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

Question 26

Sanger dideoxy sequencing

Answer 26

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

Question 27

limitations of Sanger sequencing

Answer 27

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

Question 28

exome

Answer 28

complete exon complement

Question 29

detection of large DNA segments

Answer 29

chromosome banding karyotype (>100kb)

Question 30

detection of specific large DNA segments

Answer 30

southern blot hybridization or FISH (>10kb)

Question 31

balanced translocation

Answer 31

all information is there but in a different spot

Question 32

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

Answer 32

selective or allele-specific hybridization, CGH

Question 33

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

Answer 33

CGH, PCR, DNA sequencing

Question 34

unbalanced translocation

Answer 34

missing material

Question 35

isochromosomes are examples of _

Answer 35

a balanced translocation

Question 36

paracentric inversion

Answer 36

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

Question 37

pericentric inversion

Answer 37

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

Question 38

FISH and karyotyping require _

Answer 38

metaphase cells because the chromosomes are condensed

Question 39

When can FISH be done with interphase cells?

Answer 39

if looking for duplication or loss

Question 40

comparative genome hybridization

Answer 40

used to detect copy number changes only

Question 41

karyotyping uses _

Answer 41

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

Question 42

karyotype disadvantages

Answer 42

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

Question 43

chromosome paint

Answer 43

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

Question 44

RFLP

Answer 44

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

Question 45

comparative genome hybridization (CGH)

Answer 45

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

Question 46

CGH method

Answer 46

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

Question 47

too much sample DNA in CGH

Answer 47

indicated a duplication

Question 48

too much reference DNA in CGH

Answer 48

indicates deletion

Question 49

SNPs

Answer 49

single nucleotide polymorphisms

Question 50

methods to detect SNPs

Answer 50

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

Question 51

micro-array based mutation scanning

Answer 51

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

Question 52

targeted gene sequencing

Answer 52

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

Question 53

advantages of targeted gene sequencing

Answer 53

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

Question 54

whole exome sequencing

Answer 54

all annotated exons are targeted

Question 55

methods to detect changes in cytosine methylation

Answer 55

sodium bisulfite treatment followed by PCR and DNA sequencing of products

Question 56

sodium bisulfite

Answer 56

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