Cell Biology: Unit 3 Lecture 4

Front 1

Where do restriction endonucleases cut DNA?

Back 1

Cleave phosphodiester linkages

Front 2

Where do restriction endonucleases originate from?

Back 2

Bacteria: used to protect there genomes from viral attacks.

Front 3

Why is a restriction enzyme digestion site referred to as palindromic?

Back 3

Reads the same forward and backwards.

Front 4

What are the three different types of cuts that can be produced with restriction enzymes?

Back 4

5' overhang
3' overhang
blunt

Front 5

Which restriction enzyme cuts are referred to as "sticky"?

Back 5

5' overhangs and 3' overhangs

Front 6

What enzyme can be used to seal two restriction enzyme digested ends?

Back 6

DNA Ligase

Front 7

What does the term recombinant DNA refer to?

Back 7

Any DNA that is generated by splicing together 2 or more piece of DNA.

Front 8

Restriction enzyme digestion of DNA can be used as "fingerprinting". Why?

Back 8

Digestion is predictable due to specific digestion sites, and DNA fragment sizes will be unique for each individual

Front 9

What is one method to separate DNA fragments with respect to size?

Back 9

Electrophoresis

Front 10

What allows DNA to be mobile in an electric field?

Back 10

Negative charge of phosphodiester backbone

Front 11

What are the two types of media that can be used for electrophoresis? When would each be appropriate?

Back 11

1. Agarose
2. Polyacrylamide: used to separate very small fragments (<500bp)

Front 12

What are cloning vectors?

Back 12

DNA transport molecules that are used to move cloned sequences between biological hosts and test tubes.

Front 13

What are plasmid vectors?

Back 13

Double stranded circular molecules, which occur naturally in bacterial cells (host of choice is E. coli). They are self-replicating and are small (good).

Front 14

What are 5 features of a plasmid vector?

Back 14

1. Origin of replication
2. Antibiotic resistance gene
3. Multiple cloning sequence containing restriction sites
4. Lac Z for blue/white selection
5. Small size

Front 15

What process allows E. coli to absorb plasmid vectors?

Back 15

Transformation

Front 16

How many copies of the vector are made once inside the bacteria?

Back 16

>100

Front 17

What are 3 methods to select for the recombinant plasmid?

Back 17

1. antibiotic selection
2. blue/white selection
3. size determination

Front 18

What are the two types of DNA libraries? What is each made from?

Back 18

1. Genomic Library: Entire genome cleaved by restriction enzymes and cloned into vectors

2. cDNA Library: RNA made into DNA with reverse transcriptase, then cloned into vectors.

Front 19

What are the major differences between a cDNA and genomic library?

Back 19

1. cDNA is from RNA vs. genomic is DNA
2. cDNA requires reverse transcription to make complimentary DNA strand
3. Different applications

Front 20

What is DNA hybridization?

Back 20

Single stranded DNA reforming double stranded conformation

Front 21

Hybridization can occur between different molecules. Name some combinations.

Back 21

DNA-DNA
DNA-RNA
RNA-RNA

Front 22

What are probes?

Back 22

Short single stranded DNA fragments used to detect complimentary sequences.

Front 23

How sensitive are hybridization reactions?

Back 23

Can detect one molecule within a cell.

Front 24

How selective are hybridization reactions?

Back 24

Can detect single base pair differences.

Front 25

How does stringency change the parameters of a hybridization reaction?

Back 25

Allow hybridization of strands without 100% base pair matching. Allow you to find similar fragments--not only identical fragments

Front 26

What is a DNA hybridization analysis called?

Back 26

Southern

Front 27

What does a southern analysis allow you to determine?

Back 27

Gene size
Genetic Map
Exon/intron structure

Front 28

What does a "northern" analyze?

Back 28

RNA expression

Front 29

What information can be determined from a northern analysis?

Back 29

transcript size
abundance of transcript
relative abundance of transcript

Front 30

What is the benefit of in situ hybridization?

Back 30

determines cellular expression, chromosomal location, location relative to other gene distribution of a particular DNA or RNA molecule

Front 31

What is the benefit of microarray analysis?

Back 31

Can compare 1000’s of genes at one time. Useful for comparing gene expression between different tissue types (brain vs heart) or normal vs malignant.

Front 32

What are 4 types of hybridization experiments?

Back 32

1. southern
2. northern
3. in situ
4. microarray

Front 33

How would you select a desired gene from a library?

Back 33

hybridization

Front 34

What is PCR? What is it used for?

Back 34

1. Polymerase Chain Reaction
2. Enzymatic amplification of selected regions of DNA

Front 35

What are the starting material needed for a PCR experiment?

Back 35

1. DNA
2. Primers
3. dNTPs
4. DNA polymerase (taq polymerase)
5. right reaction conditions

Front 36

PCR cycle requires 3 steps. What are they and what are the temperature for each?

Back 36

1. Denaturation--94
2. Annealing--variable
3. Extension--72

Front 37

How many cycles is a typical PCR reaction?

Back 37

20-35 cycles

Front 38

What are 3 requirements for DNA polymerase?

Back 38

Template
Primer
3' -OH

Front 39

How is the DNA chain terminated in chain termination sequencing?

Back 39

ddNTPs
does not have a 3' -OH

Front 40

What is the major limitation of DNA sequencing? How is it overcome?

Back 40

1. Can only do short fragments (<750bp)
2. Shotgun sequencing

Front 41

What is shotgun sequencing?

Back 41

This process involves breaking the genome DNA into fragments, determining the sequence of each fragment and using computer programs to search for overlaps to assemble the whole sequence.

Front 42

Why is a genome map useful in sequencing?

Back 42

provides a guide for the sequencing experiments by showing the positions of genes and other distinctive features

Front 43

What is the difference between genetic mapping and physical mapping?

Back 43

GM is based on the use of genetic techniques to construct maps showing the positions of genes on a genome. Cross-breeding experiments, examination of family histories (pedigrees).

PM uses molecular biology techniques to examine DNA molecules directly.

Front 44

What is the primer used in cDNA synthesis?

Back 44

oligo-dT
compliment to RNA poly-A tail

Front 45

What markers can be used to locate probes?

Back 45

32P
fluorescent markers

Front 46

In the 3rd cycle of PCR, how many double stranded DNA fragments do you have?

Back 46

8

Front 47

What technique can you use to amplify mRNA?

Back 47

RT-PCR
PCR must start with DNA template