Biochem Mod Iii

Front 1

A researcher is trying to identify a specific protein within a mixture. He subjects the mixture to gel electrophoresis and then transfers the gel to a nitrocellulose membrane. The membrane is incubated with a monoclonal antibody followed by a secondary antibody to visualize the protein. This technique represents:

Back 1

Western Blot

Front 2

What does northern blot measure?

Back 2

NORTHERN MEASURE RNA NITROCELLUOSE DNA PROBE

Front 3

What does southern blot measure?

Back 3

SOUTHERN MEASURES DNA NITROC ELLUOSE WITH DNA PROBE

Front 4

What does western blot measure?

Back 4

WESTERN MEASURES PROTEINS VIA ANTIBODIES GEL ELECTROPHORESIS

Front 5

What does RNAi do?

Back 5

--The RNAi blocks the translation of the VEGF mRNA.

-- The RNAi induces the degradation of VEGF mRNA.

Front 6

Tell me about the DNA chip.

Back 6

--Permits the screening of many genes simultaneously to determine which alleles of these genes are present in the sample obtained from patients

--To determine which genes are being expressed if the mRNA from a tissue specimen is used to produce cDNA by reverse transcriptase the cDNA will hybridize with only those genese being expressed in that tissue

--Used to diagnose infectious diseases

Front 7

Whats up with DNA sequencing?

Back 7

--Most common procedure for determining the sequence of nucleotides in a DNA strand.
--Synthesized from 5’-3’ end.
--Four separate reactions performed
--Read gel bottom to top.

Front 8

Whats up with restriction fragments and reading them?

Back 8

MOST RESTRICTION SITES ARE PALIDROMS ON BOTH STRANDS WHEN READ FROM 5’-3’.

Front 9

Just go ahead and tell me everything your know about restriction enzyme?

Back 9

--Enzymes called restriction endonucleases enable molecular biologist to cleave segments of DNA from the genome of various types of cells or to fragment DNA obtained from other sources
--A restriction enzyme is an endonuclease that specifically recognizes a short sequence of SNA (4-6bp) un length and cleaves a phosphodiester bond in both DNA strands within this sequene.
--Most cut palindromes(strands read the same both ways)
--Restriction enzymes were originally used in bacteria to limit the growth of viruses
--Restriction fragments can be used to identify variations in base sequence in a gene.
--Can be used to synthesize a recombinant DNA which is composed of molecules of DNA from different sources that have been recombined in vitro.
--Sticky ends join together. The restriction enzyme cuts the DNA and then they recombinate.

Front 10

Tell me about DNA produced from reverse transcriptase?

Back 10

--mRNA transcribed from a gene is isolated and used as a template by the enzyme reverse transcriptase which produces a DNA (cDNA) copy of the RNA.
--DNA produced by reverse transcripates does not contain inrons because mRNA has no introns.
--cDNA also lacks the regulatory regions of a gene as those sequences are not transcribed.

Front 11

Chemical synthsis of DNA?

Back 11

--Automated machines can synthesize oligonucleotides up to 250 nucleotides in length.
--Oligonucleotides can be used in the process of identifying, isolating, and amplifying genes
--Oligonucleotides can be stitched together to produce full size genes.
--Probes are single stranded polynucleotide of DNA or RNA that is used to identify a complementary sequence of a larger single –stranded DNA or RNA molecule.
--Formation of base pairs with a complementary strand is called annealing or hybridization.
--Probes can be formed from cDNA, fragments of genomic DNA, chemically syntheszizd oglionucleotides or RNA.
--Stringency refers to how exact a match the probe mush have to the DNA to which it is hybridized in order for significant hybridization to occur
--High stringency requires an exact match of BP.
--Probes carry a label

Front 12

Gel electrophorsis?

Back 12

--Use an electrical field to separate molecules on the basis of size. The negatively charge phosphate group on DNA allows it to be attracted to the cathode.
--Different gels are used depending on the size of the DNA
--Bands can be viewed by different staining methods

Front 13

Detection of specfic DNA sequences?

Back 13

--DNA is transferred to a solid support
--Southern blot is used to identify DNA sequences on gels.
--Produced when DNA on a nitrocellulose blot of an electrophoretic gel is hybridized with a DNA probe
--Northern blots are produced when RNA on a nitrocellulose blot is hybridized with a DNA probe
--Westblot involves separating proteins by gel electrophoresis and pprobing with labeled antibodies for specific proteins
--DNA sequencing the most common procedure for determining the seuquence of nucleotides in a DNA strand. And involves the use of dideoxynucleotides

Front 14

Where are most DNA polymorphism present?

Back 14

o Most are present in the noncoding regions

Front 15

How do you detect polymorphism?

Back 15

Restriction fragment length polymorphisms

Front 16

Whats up with cloaning DNA?

Back 16

--The first technique from amplifying the quantity of DNA is know as cloaning.
--DNA is attached to a vector and this is introduced into the host cell that makes multiple copies of the DNA.

Front 17

2. PCR (polymerase chain reaction) was a fundamental discovery in the field of molecular biology, but the most important discovery within PCR was the discovery of Taq polymerase isolated from a thermophilic bacterium (Thermus aquaticus) found in hot springs. Choose the statement that is the most accurate in describing the function of Taq polymerase.

A. The Taq polymerase protein is heat stable
B. Any polymerase isolated from any bacteria will work in PCR
C. Taq polymerase is less heat stable
D. Taq polymerase has a higher processivity then other polymerases
E. Taq polymerase makes more mistakes then most bacteria .

Back 17

The Taq polymerase protein is heat stable

Front 18

. Which of the following sequences is a potential restriction site?

A. CATATG
B. CATTAC
C. CATCAT
D. GGGCCA
E. AAAAAA
AND WHY?

Back 18

A. CATATG

Front 19

An 18-year-girl is found dead in her apartment. A forensic pathologist removes a hair found at the crime scene, isolates DNA from the hair and replicates it exponentially. This method is referred to as:

A. gene expression.
B. genotyping.
C. polymerase chain reaction.
D. Single nucleotide polymorphism.
E. DNA mapping.

Back 19

C) PCR

Front 20

5. A 22-year-old female and a 23-year-old male are expecting their first child. The female is a carrier of the sickle cell trait, because her mother had sickle cell disease. On their visit to a genetic counselor a sample of fetal DNA is obtained through an amniocentesis. In order to ascertain the status of the fetus, which one of the following laboratory test would be used to determine if the fetus has the sickle cell mutation?

A. DNA sequencing
B. Northern Analysis
C. Hemoglobin Electrophoresis
D. Western Blot
E. RNAi

Back 20

A. DNA sequencing

Front 21

The best method to measure albumin mRNA in a hepatocarcinoma cell line is a:

A. Eastern Blot
B. Southern Blot.
C. Northern Blot.
D. Western Blot

Back 21

C. Northern Blot.

Front 22

. Which of the following statements correctly describes a feature of DNA migration within a gel?

A. Larger DNA fragments migrate further in the gel than smaller DNA fragments
B. DNA fragments migrate toward the negative electrode (anode)
C. DNA fragments migrate toward the positive electrode (cathode)
D. RNA fragments migrate toward the positive electrode (cathode)
E. RNA fragments migrate toward the negative electrode (anode)

Back 22

C. DNA fragments migrate toward the positive electrode (cathode)

Front 23

A 65-year-old patient with macular degeneration is having trouble with her eyesight and it is getting to the point of impacting her daily activities. The doctor recommends a new study involving RNAi for the treatment of macular degeneration. The target for this new drug is VEGF, which is overexpressed in macular degeneration. How would you explain to the patient how this new drug works?

A. The RNAi blocks the translation of the VEGF mRNA.
B. The RNAi blocks the transcription of the VEGF gene.
C. The RNAi induces the degradation of VEGF mRNA.
D. The RNAi inhibits VEGF mRNA transport.
E. The RNAi blocks VEGF export.

Back 23

A. The RNAi blocks the translation of the VEGF mRNA

C. The RNAi induces the degradation of VEGF mRNA.

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