Cell Biology For Bme: Chapters 10 - Analyzing Genes And Genomes

Front 1

When did it become possible for the first time to isolate a given piece of DNA?

Back 1

1970s

Front 2

What is Recombinant DNA Technology?

Back 2

Collection of techniques by which DNA segments from different sources are combined to make new DNA.

Front 3

Recombinant DNAs are widely used in what?

Back 3

Cloning of genes and molecular biology generally.

Front 4

Recombinant DNA Technology makes it possible to do what (2 things)?

Back 4

To create chromosomes with combinations of genes that could never have formed naturally or ones that could happen but would take thousands of years.

Front 5

What are the two methods for isolating and producing DNA sequences in large numbers?

Back 5

1. DNA cloning
2. Polymerase Chain Reaction (PCR)

Front 6

What does a Nuclease do?

Back 6

It catalyzes the hydrolysis of phosphodiester bond in a nucleic acid.

Front 7

What are Restriction Nucleases?

Back 7

They are nucleases that cut double-stranded DNA at specific sites determined by short sequences of nucleotide pairs.

Front 8

How is the bacterium's own DNA protected from cleavage by restriction nucleases?

Back 8

It is protected from cleavage by chemical modification of the same sequences used by the nuclease.

Front 9

TRUE OR FALSE: Different bacterial species contain the same restriction nucleases that cut at the same, general sequence of nucleotides.

Back 9

FALSE. Different bacterial species contain DIFFERENT restriction nucleases that each cut at a DIFFERENT, SPECIFIC sequence of nucleotides.

Front 10

Regarding restriction nucleases, the target sequences are often what in terms of ordering?

Back 10

Palindromic! (Nucleotide sequence is symmetrical around a central point)

Front 11

Regarding restriction nucleases, some nucleases cut the strand staggered. What do these staggered cuts generate?

Back 11

They generate "sticky-ends" (short, single-stranded DNA overhangs) that help the cut DNA join back together through complementary base-pairing.

Front 12

Where do restriction nucleases come from?

Back 12

They are obtained from bacteria!

Front 13

What separates DNA fragments of different sizes?

Back 13

Gel Electrophoresis!

Front 14

Gel electrophoresis separates DNA fragments on what basis?

Back 14

Their length!

Front 15

Is DNA positively or negatively charged?

Back 15

NEGATIVELY charged!

Front 16

Do larger fragments of DNA move faster or slower in gel electrophoresis? Why?

Back 16

SLOWER because their progress is more impeded by the agarose gel matrix (small pores)

Front 17

How do you isolate a particular DNA fragment from gel electrophoresis?

Back 17

It can be cut out using a scalpel or razor blade and the specific DNA can then be extracted

Front 18

TRUE OR FALSE. DNA bands on agarose or polyacrylamide gels are invisible unless the DNA is labeled or stained in some way.

Back 18

TRUE.

Front 19

What are two ways to label DNA so it can be seen in electrophoresis?

Back 19

1. Dyeing it with a florescent dye to be seen under UV light
2. Incorporating a radioisotope into the DNA before electrophoresis like P^32

Front 20

How does one identify a fragment that has a specific nucleotide sequence?

Back 20

Hybridization!

Front 21

What is Hybridization?

Back 21

Experimental process in which two complementary nucleic acid strands form a double helix; a powerful technique for detecting specific nucleotide sequences.

Front 22

What is a DNA probe?

Back 22

Single-stranded DNA molecule with 10-1000 nucleotides used in hybridization to detect specific nucleic acids with complementary code.

Front 23

Resulting hybridization using DNA probes can be visualized by using what?

Back 23

Southern Blotting!

Front 24

What are the 5 basic steps to Southern Blotting?

Back 24

1. Electrophoresis
2. Separated DNA fragments in gel is blotted onto nitrocellulose or nylon paper laying on sponge and alkali solution
3. Nitrocellulose sheet is peeled off
4. Sheet is exposed to radioactively labeled DNA probes specific for the required DNA sequence
5. Sheet is washed well so that only hybridized probe molecules remain attached (which then can be visualized using autoradiograph)

Front 25

How are separated DNA fragments blotted onto nitrocellulose paper?

Back 25

Alkali solution under gel (buffer) is sucked through gel which denatures the DNA and transfers the single-stranded fragments from the gel to the surface of the sheet.

Front 26

What is Northern Blotting?

Back 26

Basically the same procedure of Southern blotting but with specific sequences of RNA.

Front 27

What joins DNA fragments together to produce a recombinant DNA molecule?

Back 27

DNA ligase!

Front 28

What is Recombinant DNA?

Back 28

Any DNA molecule constructed in the laboratory.

Front 29

What is Transformation?

Back 29

Mechanism that introduces DNA into bacteria from their surrounding.

Front 30

In transformation, incoming DNA is often incorporated into the bacterial genome by what?

Back 30

Homologous recombination

Front 31

What two things are needed to combine two complementary staggered ends of DNA?

Back 31

DNA ligase and ATP!

Front 32

What four things are needed to combine a blunt end with a staggered end of DNA?

Back 32

First: 1. Deoxyribo-nucleotides and
2. Polymerase (to fix the staggered end)
Then: 3. Ligase
4. ATP

Front 33

To maintain foreign DNA in a bacterial cell, what is used as a carrier or vector?

Back 33

Bacterial Plasmid!

Front 34

What does a plasmid vector containing a replication origin enable it to do?

Back 34

It enables it to replicate in the bacterial cell independently of the bacterial chromosome!

Front 35

What 3 things do plasmids contain that make them awesome for cloning?

Back 35

1. Their own replication origin
2. Cleavage sites for restriction nucleases
3. Genes for some selectable property (like antibiotic resistance)

Front 36

How do you insert a piece of DNA into a plasmid?

Back 36

Purified plasmid DNA is exposed to restriction nucleases that cleaves it in one place, then the DNA fragment to be cloned is inserted into it by DNA ligase. This plasmid is then introduced to the bacterium by transformation where it doubles in number every 30 minutes.

Front 37

What is a DNA Library?

Back 37

The collection of cloned DNA fragments in the resulting bacterial culture.

Front 38

What is a Genomic Library?

Back 38

The collection of DNA fragments that are derived directly from the chromosomal DNA.

Front 39

What is cDNA?

Back 39

Complementary DNA is a DNA molecule made as a copy of mRNA and therefore lacking the introns present in genomic DNA.

Front 40

What are cDNA Libraries?

Back 40

Libraries that represent the mRNA produced by a particular tissue!

Front 41

Because patterns of gene expression charge during development, what happens to cDNA libraries?

Back 41

cDNA libraries will also change to reflect the genes expressed by cells at different developments!

Front 42

What is the most important advantage of cDNA?

Back 42

cDNA clones contain the uninterrupted coding sequence of the gene (unlike genomic libraries)

Front 43

If the aim of cloning the gene is either to deduce the amino acid sequence of the protein from the DNA or to produce the protein in bulk, what type of DNA library should be used?

Back 43

cDNA!

Front 44

What is the main advantage of genomic clones?

Back 44

They contain introns as well as exons and they include the regulatory sequences that determine when and where genes are expressed.

Front 45

Genomic clones are used to determine what?

Back 45

The complete nucleotide sequences of genomes!

Front 46

When was PCR invented?

Back 46

1980s

Front 47

What is PCR?

Back 47

Polymerase Chain Reaction is a technique for amplifying specific regions of DNA by multiple cycles of DNA synthesis each followed by a brief heat treatment to separate complementary DNA strands.

Front 48

TRUE OR FALSE: PCR can be carried out entirely in vitro.

Back 48

TRUE!

Front 49

What does the first cycle of PCR produce?

Back 49

Produces two double-stranded DNA molecules!

Front 50

In PCR, what direct the amplification of the desired piece of DNA?

Back 50

PCR primers!

Front 51

What are the 5 steps to the first cycle of amplification in PCR?

Back 51

1. Heat to separate strands
2. Cool and add primers
3. Hybridization of Primers
4. Add polymerase and transcription factors
5. DNA synthesis from primers

Front 52

PCR depends on the use of what?

Back 52

The use of a special DNA polymerase isolated from a thermophilic bacterium (it has to be able to withstand the heat treatment!)

Front 53

Assuming you start with one double stranded fragment of DNA, what are the 3 cycles of PCR?

Back 53

1. Production of two double stranded DNA molecules
2. Production of four double-stranded DNA molecules
3. Production of eight double-stranded DNA molecules
...which goes on and on until the demanded quantity of DNA is present!

Front 54

In practice, how many cycles of PCR are required for useful amylification? How long does each cycle take?

Back 54

20-30 cycles are needed and each cycle is about 5 minutes long!

Front 55

What are 4 super useful applications of PCR?

Back 55

1. Easy cloning of small (under 10K nucleotides) DNA fragments
2. Can be used to clone DNA or RNA (think lots of genomic copies or cDNA copies!)
3. Detection of infections by pathogens at very early stages
4. Forensic medicine

Front 56

What is Dideoxy DNA sequencing?

Back 56

A method based on DNA synthesis carried out in vitro in the presence of a special, chain-terminating dideoxyribonucleoside triphosphates.

Front 57

In dideoxy DNA sequencing, DNA replication reactions are performed under conditions that ensure what?

Back 57

Ensure that the new DNA strands terminate when a given nucleotide (A, G, C, or T) is reached.

Front 58

Dideoxyribonucleoside triphosphates differ from normal deoxyrimonucleoside triphosphates in what way?

Back 58

They lax the 3' hydroxyl group

Front 59

What are Expression Vectors?

Back 59

Vectors that produce high-levels of a protein because they include the appropriate transcription and translation signals.

Front 60

Because the protein encoded by the expression vector is typically produced inside the cell, it must by purified from the host cell proteins after cell lysis by what?

Back 60

Chromatography!

Front 61

What was the first organism to have its complete genome sequence determined?

Back 61

Infectious bacterium Haemophilus Influenzae

Front 62

What can effectively disrupt the shotgun approach to DNA sequencing?

Back 62

Large repetitive nucleotide sequences!

Front 63

Clues to a protein's function can be obtained by examining what?

Back 63

When and where its gene is expressed in the cell, or in the organism as a whole.

Front 64

Determining the pattern and timing of a gene's expression can be accomplished by joining the regulatory region of the gene under study to what kind of gene?

Back 64

A reporter gene!

Front 65

How is the expression of a reporter gene monitored?

Back 65

By tracking the fluorescence or enzymatic activity of its protein product.

Front 66

What is one of the most popular reporter proteins used today?

Back 66

Green Fluorescent Protein (GFP)

Front 67

What is GFP?

Back 67

Green Fluorescent Protein is a molecule used as a reporter protein that gives luminescent jellyfish their greenish glow.

Front 68

How is GFP added to a protein in question?

Back 68

Usually it can simply be attached to the end of the gene that encodes a protein of interest.

Front 69

What is in situ hybridization?

Back 69

Technique that allows specific nucleic acid sequences to be located while they are still in place within the cells/chromosomes.

Front 70

What is a DNA microarray?

Back 70

Tool that allows the RNA products of tens of thousands of genes to be monitored at the same time.

Front 71

What is the purpose of DNA microarray?

Back 71

By examining the expression of so many genes at the same time, we can begin to identify and study the complex gene expression patters that underlie cell physiology.

Front 72

What is Site-Directed Mutagenesis?

Back 72

Technique by which a mutation can be made at a particular site in DNA.

Front 73

Site-Directed Mutagenesis allows one to determine what 2 things?

Back 73

1. Which parts of the polypeptide chain are crucial to fundamental processes
2. the biological roles of each part of a given protein

Front 74

What is Gene Replacement?

Back 74

The replacement of a normal gene in an organism with one that has been mutated in vitro

Front 75

How can Gene Replacement be accomplished?

Back 75

By homologous recombination between the introduced mutant DNA and the chromosomal DNA.

Front 76

What is a Gene Knockout?

Back 76

A gene is deleted entirely on purpose

Front 77

What are Transgenic Organisms?

Back 77

Also called Genetically Modified Organisms (GMOs) are organisms that have had genes added or altered to their genomes.

Front 78

What is RNA interference?

Back 78

RNAi is a cellular mechanism activated by double-stranded RNA molecules that results in the destruction of RNAs containing as imilar nucleotide sequence.

Front 79

RNAi is widely exploited to do what?

Back 79

Exploited as an experimental tool for preventing the expression of selected genes.

Front 80

In regards to preventing the expression of selected genes, how does RNAi technique work?

Back 80

It works by introducing the cell a foreign but matching piece of RNA to the gene to be inactivated. This tricks the cell into degrading BOTH sequences.

Front 81

Can RNAi produce heritable changes in gene expression?

Back 81

YES