Topic 15: Molecular Techniques

Question 1

What is nucleic acid hybridization?

Answer 1

Base pairing of one strand of a nucleic acid to a complementary sequence on a strand from a different nucleic acid molecule

Question 2

What is DNA sequencing used for?

Answer 2

Using the principle of complementary base pairing to determine the complete nucleotide sequence of DNA molecule

Question 3

What do restriction enzymes do?

Answer 3

Cut DNA molecules at a limited number of specific locations

Question 4

What is gel electrophoresis?

Answer 4

Separate out a mixture of nucleic acid fragments by size, charge or other physical properties using a gel made of a polymer as a molecular sieve

Question 5

What is the purpose of PCR?

Answer 5

Obtain many copies of a desired gene

Question 6

What are the 3 steps of PCR?

Answer 6

1. Desaturation: heat briefly to separate DNA strands
2. Annealing: cool to allow primers to form hydrogen bonds with ends of target sequence
3. Extension: DNA polymerase adds nucleotides to the 3’ end of each primer

Question 7

What is a nucleic acid probe?

Answer 7

A short, single stranded nucleic acid that can be either RNA or DNA used to detect the mRNA by nucleic acid hybridization, labeled with a fluorescent tag

Question 8

What does FISH stand for?

Answer 8

Fluorescent in situ hybridization

Question 9

What is fluorescent in situ hybridization?

Answer 9

Using a nucleic acid probe to hybridize specifically with any complementary sequences on the many mRNAs in cells where the gene is being transcribed

Question 10

What is the purpose of FISH?

Answer 10

Determine where single genes are expressed by looking at mRNA

Question 11

What does RT-PCR stand for?

Answer 11

Reverse transcriptase polymerase chain reaction

Question 12

What are the 5 steals of making cDNA from mRNA?

Answer 12

1. Reverse transcriptase added to mRNA
2. Reverse transcriptase makes first DNA strand using mRNA as template
3. mRNA degraded
4. DNA polymerase synthesizes second DNA strand
5. Results in cDNA

Question 13

What is cDNA?

Answer 13

Complementary DNA made from mRNA that lacks introns

Question 14

What are the 3 steps of RT-PCR?

Answer 14

1. cDNA synthesis
2. PCR amplification
3. Gel electrophoresis

Question 15

What is the purpose of RT-PCR?

Answer 15

Compare gene expression between samples

Question 16

What is a DNA microarray?

Answer 16

Tiny amounts of large number of single-stranded DNA fragments representing different genes fixed to a glass slide in a grid of dots

Question 17

What is the purpose of DNA microarray assays?

Answer 17

Analyze expression of many genes

Question 18

What are the 6 steps of a microarray assay?

Answer 18

1. Isolate mRNA from normal cell and tumor cell
2. Prepare cDNA w fluorescent tag
3. Separate cDNA into single strands and add to microarray
4. Fluorescent strands anneal to complementary sequences
5. Wash unhybridized cDNA strands
6. See color

Question 19

What is RNA sequencing?

Answer 19

Sequence the cDNA samples from different tissues in order to discover which genes are expressed

Question 20

What are the 5 steps of RNA sequencing?

Answer 20

1. Isolate mRNA samples
2. Cut into shorter fragments
3. Convert into cDNAs
4. Sequence cDNA
5. Map cDNA onto genome of species in question

Question 21

What is Sanger sequencing?

Answer 21

Using chain-terminating dideoxynucleotides during DNA replication

Question 22

What is SDS-PAGE used for?

Answer 22

Separate proteins on the basis of their size or charge

Question 23

How does SDS-PAGE work?

Answer 23

Using gel electrophoresis, proteins will move to anode or cathodes, then we can identify the presence of separated proteins

Question 24

What is Southern Blotting used for?

Answer 24

Detect a specific DNA sequence in a sample

Question 25

What is a chromosome painting used for?

Answer 25

Compare different chromosomes present between a abnormal and normal sample