Biochem Block 2: Recombinant Dna 2

Front 1

How do you remember what assays are used for DNA, protein or RNA?

Back 1

SNOW DROP

Front 2

With Southern blotting, what kind of electrophoresis gel do you use?

Back 2

agarose

Front 3

With Southern blotting, the fragment is denatured using...

Back 3

a strong alkali solution

Front 4

Southern blotting: After DNA fragments are denatured into single strands with alkali, they are transferred onto a...

Back 4

nitrocellulose or nylon sheet

Front 5

Southern blotting: Once on a nitrocellulose or nylon sheet, ssDNA fragments are...

Back 5

hybridized with a probe

Front 6

The Northern, Southern and Western blots all follow what 5 steps?

Back 6

1. Fragmentation
2. Separation
3. Denaturation
4. Blotting
5. Hybridization w/probe

Frank Sat Down By Hannah

Front 7

What kind of probes are used in the 3 blotting techniques?

Back 7

Southern (DNA) and Northern (RNA) = DNA probe

Western (protein) = Antibody probe

Front 8

DNA is denatured before or after transferring (blotting) it onto a nitrocellulose paper?

Back 8

before

Front 9

What is needed for DNA sequencing?

Back 9

4 tubes

ONE dideoxynucleotide (ddATP, ddTTP, ddGTP, or ddCTP) per tube

four normal deoxynucleotides (dATP, dGTP, ...) in each tube

DNA polymerase in each tube

Primer in each tube

Single stranded DNA template in each tube

Magnesium to shield DNA's negative charge

Front 10

What is a dideoxynucleotide?

Back 10

it's the same as a DNA nucleotide except it lacks the hydroxyl group; therefore you cannot elongate a DNA chain with it

Front 11

Using DNA sequencing, nucleotide synthesis terminates when a __________ is incorporated into the growing chain.

Back 11

ddNTP

Front 12

Describe the Sanger Method

Back 12

Front 13

How can we amplify specific DNA sequences?

Back 13

1. DNA cloning
2. Libraries
3. PCR

Front 14

A bacteria is transformed while a eukaryote is ______________ when you insert a DNA sequence into the genome.

Back 14

transfected

Front 15

When you insert a sequence into a host like a bacteria and allow it to grow and divide naturally to amplify the DNA, this method is called...

Back 15

DNA cloning

Front 16

With a bacteria host, the 3 possible vectors include...

Back 16

1. bacteriophage
2. plasmids
3. cosmids - genetically engineered like plasmid but bigger

NOTE: A vector is used to insert the sequence into DNA

Front 17

With a eukaryotic host, the 4 possible vectors include...

Back 17

1. retroviruses
2. adenoviruses
3. free DNA
4. DNA coated w/lipid bilayer (liposome)

Front 18

The genomic library has (larger/small) fragments than cDNA. The cDNA library has (larger/smaller) fragments.

Back 18

larger

smaller

Front 19

Rank the capacity of these vectors for foreign DNA for the genomic library:
1. Bacteriophage
2. BACs (engineered bacterial DNA)
3. YACs (engineered yeast DNA)

Back 19

bacteriophage < BACs < YACs (largest)

Front 20

What is the vector of choice for foreign DNA for the cDNA library? How is the cDNA library obtained? cDNA corresponds to _______________________.

Back 20

plasmid

cDNA is obtained from mRNA using reverse transcriptase.

corresponds to a specific stage of differentiation of the cell

Front 21

Which library corresponds to a specific stage of differentiation of a cell?

Back 21

cDNA library

Front 22

PCR requires:

Back 22

1. isolated DNA
2. 2 types of primers that are not complementary to each other w/NO TANDEM REPEATS
3. 4 dNTPs
4. Heat stable DNA polymerase (because you will heat DNA to open it)
5. Magnesium to protect the negative charge of DNA

Front 23

DNA polymorphisms are...

Back 23

variations of DNA within the same species (hair color)

Front 24

Polymorphisms result from (3):

Back 24

1. point mutations
2. deletions
3. insertions

Front 25

What techniques can be used to detect polymorphisms (5)?

Back 25

1.Restriction fragment length polymprophisms (RFLPs)
2. Detection of mutations by allele-specific oligonucleotide probes
3. testing for mutation by PCR
4. Detection of polymorphisms caused by repetitive DNA
5. DNA chips/DNA microarray

Front 26

Detection of polymorphisms: RFLPs?

Back 26

DNA is cut by restriction enzymes, which cut at specific sites. If there are fragments that are larger or smaller than others, this is due to a mutation that abolishes the normal restriction site. These fragments with different lengths are called restriction fragment length polymorphisms (RFLPs).

Front 27

RFLPs can be used for genetic testing because mutations causing diseases affect a restriction site in what 2 ways?

Back 27

in a coding region

outside of a coding region but still associated with the abnormal gene causing the disease

Front 28

RFLPs: the mutation that causes sickle cell anemia abolishes a restriction site for the enzyme ___________ in the __________ gene, making a RFLP that is larger than the RFLP for a normal individual.

Back 28

-Enzyme: Mstll
-Gene: Beta-globin

Front 29

What can oligonucleotide probes be used to identify mutations?

Back 29

Two probes: one is complementary to sequence in normal allele, another is complementary to sequence in mutated allele. Sequence of interest amplified by PCR and then DOT BLOTTED onto nitrocellulose paper. Paper is treated with radioactive probe (32P). Incubated and washed; XRAY film made. Analysis of xray: shows if allele of interest is normal or mutated; for carriers, both normal and mutated alleles will be detected; for affected pts, only mutant probe will hybridize.

Front 30

When you detect mutations by allele specific oligonucleotide probes, the xray film of a carrier will show...

Back 30

both normal and mutated alleles will hybridize

Front 31

When you detect mutations by allele specific oligonucleotide probes, the xray film of an affected pt will show...

Back 31

only the mutant probe will hybridize

Front 32

In addition to DNA sequencing, ______________ can be used to detect cystic fibrosis.

Back 32

allele-specific oligonucleotide probes

Front 33

When testing for cystic fibrosis using oligonucleotide probes, one probe binds to the mutant _________ gene while the other binds to the ___________ gene.

Back 33

-mutant deltaF508 gene
-normal gene

Front 34

When a sequence containing a mutation is used as a primer for PCR, normal DNA (will/will not) hybridze with the primer. Will amplification take place?

Back 34

will not

no amplification

Front 35

_____________ can be used as primes to test clinically for several mutations in a short time.

Back 35

Oligonucleotides for different mutations

Front 36

Polymorphisms can be detected due to repetitive DNA because the human genome has __________________. These ___________ can be digested by restriction enzymes and the fragment sizes differ in size from one individual to another.

Back 36

variable number tandem repeats (VNTR)

Front 37

VNTR patterns can be used to ______________.

Back 37

identify individuals/family relationships/forensic analysis