Unit 2 - 10. Molecular Techniques

Question 1

Recombinant DNA

Answer 1

• formed by joining DNA molecules from different sources

Question 2

Gene cloning

Answer 2

• produces exact copies of DNA fragment by inserting the fragment into a self-replicating vector which is incorporated into cultured host cells

Question 3

Genomic library

Answer 3

• contain overlapping DNA fragments that represent the entire genome of an organism

Question 4

cDNA Library

Answer 4

• generated from mRNA and represent exons from genes that are expressed by particular cell

Question 5

hybridization

Answer 5

• the joining together of two complementary strands of nucleic acid to form a double stranded, hybrid molecule

Question 6

palindrome

Answer 6

• word/phrase/sentence read the same backwards as forwards

Question 7

probe

Answer 7

• a labeled nucleic acid used to identify a complementary region in a clone or genome

Question 8

Vectors used in gene cloning

Answer 8

1. Plasmids
2. Bacteriophages
3. Cosmids
4. Bacterial Artificial Chromosome
5. Yeast Artificial Chromosome

Question 9

1. Plasmids

Answer 9

• insert up to 15 kb

- circular double-strand DNA molecules that replicate outside of main chromosome (bacteria/yeast)

Question 10

2. Bacteriophage

Answer 10

• 20 kb

- viruses that infect bacteria

Question 11

3. Cosmids

Answer 11

• 45 kb

- modified plasmids that can hold longer pieces of DNA

Question 12

4. Bacterial Artificial Chromosomes (BAC)

Answer 12

• 100-300 kb

- a very long DNA plasmid modified to carry DNA fragment

Question 13

5. Yeast Artificial Chromosome (YAC)

Answer 13

• 100-2000 kb

- genetically modified yeast chromosome that can accept very large DNA fragments

Question 14

Restriction Endonucleases

Answer 14

• enzymes that cut DNA at short specific nucleotide sequences (usually palindromes) used in recombinant DNA technology to cut DNA into smaller fragments
• Ex: E.coli enzyme - EcoR1

Question 15

Steps in Gene Cloning

Answer 15

1. Isolate DNA using restriction enzyme
2. Treat appropriate vector with same restriction enzyme
3. Sticky ends make recombinant DNA with fragment and vector
4. replicate by transferring to bacterial or yeast cell

Question 16

Uses in Gene Cloning

Answer 16

• used to make gene library for gene mapping
• produce large quantities of protein product (insulin)
• use as probes to detect complementary nucleic acids
• used in gene therapy

Question 17

Polymerase Chain Reaction (PCR)

Answer 17

• mimics in vivo DNA replication = used as alternative to gene cloning to amplify a specific DNA sequence
• requires primers, DNA polymerase, deoxynucleotide triphosphate

Question 18

Steps in PCR

Answer 18

1. Denaturation - separate ds DNA into ss DNA by heat (95 Celsius)
2. Annealing - reaction is cooled (50-65 Celsius) to allow primers to bind to DNA
3. Extension - use Taq polymerase (thermostable DNA polymerase), dNTPs, and heat (75 Celsius) to lengthen sequence
   Repeat steps 1-3 to generate millions of copies

Question 19

Uses of PCR

Answer 19

• rapid, sensitive detection of pathogens (TB, HIV)
• analysis of cells for genetic mutation (sickle cell anemia)
• DNA analysis in forensic, evolutionary studies

Question 20

RT-PCR

Answer 20

• reverse transcription polymerase chain reaction

- used to amplify DNA from RNA source

Question 21

Quantitative (Q) PCR

Answer 21

• uses internal control to calculate amount of nucleic acid in original sample

Question 22

Real Time PCR

Answer 22

• a faster method of target amplification and detection steps in same tube
• uses fluorescent probes

Question 23

DNA Sequencing

Answer 23

• uses dideoxynuclotide triphosphates with fluorescent tags to create DNA chains of varying length
• DNA fragments are analyzed by electrophoresis and automatic scanner

Question 24

Next (2nd) generation sequencing

Answer 24

• simultaneous amplification and sequencing of many DNA fragments throughout the genome

Question 25

Uses of DNA Sequencing

Answer 25

- detecting individual mutations - discovering new genes - designing probes and PCR primers - Human genome project

Question 26

In situ hybridization

Answer 26

- uses labeled DNA or RNA probe to detect a complementary DNA sequence in cells, tissue, or chromosomes

Question 27

Steps in Southern Blotting

Answer 27

1. Isolate DNA and digest with restriction enzymes 2. Separate fragments via gel electrophoresis 3. Transfer/blot fragments to nitrocellulose paper 4. Add labeled probe to identify specific DNA fragments via hybridization (wash off excess probe) 5. Examine bands = fragments containing gene of interest

Question 28

Uses in Southern Blotting

Answer 28

- identify pattern of DNA fragments created by treatment of DNA with restriction enzymes - compare band patterns of normal and diseased individuals

Question 29

DNA microarrays (DNA Chips)

Answer 29

- uses thousands of known DNA sequences and hybridization to identify a gene sequence level of gene expression - mRNA reverse transcribed into cDNA with labeled florescent dye is stimulated by a laser - expression analyzed via red, green, and yellow spots

Question 30

Uses of DNA microarrays

Answer 30

- discovering functions of genes - diagnosis and monitoring of diseases - monitoring responses of cells to drugs