BPS 3101 Lecture Notes - Lecture 3: Ethidium Bromide, In Situ Hybridization, Antisense Rna

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champagnepanda261

29 Aug 2016

School

University of Ottawa

Department

Biopharmaceutical sciences

Course

BPS 3101

Professor

Linda Bonen

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Document Summary

Topic 3: techniques and tools for studying dna. Restriction enzyme cleavage & agarose gel electrophoresis: restriction enzymes: cleave dna into specific, small fragments, can form blind ends and sticky ends, steps, 1. Dna endonucleases cut double-stranded dna at specific recognition sites (often palindromic: 2. Recognition sequences often 4 or 6 bp can be useful for generating very large fragments in genomic mapping: 3. Two different restriction enzymes may generate the same sticky ends : compatible sticky ends are useful for cloning, 4. Isoschizomers restriction enzymes with identical recognition sequences: but may have different response to methylation states, ex. Msp1 cleaves 5"ccgg3" regardless of methylation, hpaii does not cleave. Isoschizomers to assess dna methylation state of genes in cancer patients: used assay called hpaii tiny fragment enrichment by ligation-mediated pcr . Help: found significant aberrancy in promoter methylation patterns compared with normal b cells, looks at ratio between hpaii/mspi, hyper-methylated genes won"t be cut with.

Related Questions

A-Which of the following is NOT one of the steps required toclone a piece of DNA using a plasmid vector?
1. Cut a vector and a DNA insert with aspecific restriction enzyme to create complementary "stickyends."
2. Isolate DNA fragments by gelelectrophoresis.
3. Ligate insert DNA and a vector withsimilar sticky ends together.
4. Transform ligated pieces of DNA into E.coli.
5. Select the plasmid vector that hasreplaced its origin of replication with insert DNA.

B-A representative genomic library of anorganism's DNA fragments results from the use of
1. DNA ligase joining multiple types ofvectors.
2. partial digestion with frequentlycutting restriction endonucleases.
3. many pieces of recombinant DNA fromseveral different types of organisms.
4. viruses that cleave the DNA into randompieces.

C-One of the most usefulmethods for identifying a specific DNA sequence is
1. the Southern blot.
2. the Western blot.
3. magnetic resonance imaging.
4. the Eastern blot.
5. thin-layerchromatography.

peachdinosaur267

1.) A restriction recognition site is repeated 1 times on a circular DNA molecule. If you treat this DNA molecule with this restriction enzyme, how many DNA fragments will you get?

a. 1

b. 2

c. 3

d. Restriction enzyme does not cut a circular molecule

2.) In an agarose gel DNA electrophoresis, the DNA fragments are separated based on their

a. Size

b. Electrical charge

c. Nucleotides sequences

d. Density

e. All of the above

3.) DNA molecules are usually separated using agarose gel rather than polyacrylamide because:

a. Agarose polymerizes faster than acrylamide gel

b. Polyacrylamide gel has pore size larger than the size of DNA molecules

c. Agarose gel do not have pores on tis matrix and can hold large DNA molecules

d. Agarose gel has larger pore size similar to the size of DNA molecules

4.) Restriction endonucleases

a. Randomly recognize and cut DNA sequences

b. Hydrolysis the phosphodiester bond of DNA backbone

c. Recognize specific sequence on double stranded DNA

d. A and B

e. B and C

taupefrog573

1) Which of the following is FALSE about plasmid vectors ued in recombinant DNA research?
A) Usually carrry an antibiotic resistance gene
B) Clonin sites are always located between genes, that is, outside coding sequences
C) May hae a multiple clonin gsite which can be cut by several restriction enzymes
D) May be expression vectors

2) Which of the following is FLASE about Sanger Dideoxy DNA sequencing protocols?
A) Different specfiic chemical reactions break the DNA at each base.
B) Fluorescent molecules can be used to detect the DNA
C) The Template is single stranded DNA
D) Many steps can be automated

3) A southern blot transfers ___ to a Nitrocellulose membrane.
A) Protein
B) RNA
C) DNA
D) Lipid

4) Which of the following is true about cDNA?
A) It is made from DNA template
B) It is isolated from bacteria
C) Reverse transcriptase is used to synthesize cDNA
D) It has introns

5) The restriction endonucleases used in recombinant DNA work:
A) Always produce blunt ends (No unpaired bases)
B) Recognize sequences 4-6 bp long
C) Cut the DNA outisde the recognition sequence
D) All of the above

S17

THANK YOU VERY MUCH!!!

BPS 3101 Lecture Notes - Lecture 3: Ethidium Bromide, In Situ Hybridization, Antisense Rna

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