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BIOL10005 Lecture Notes - Lecture 11: Dna Ligase, Dna Replication, Electrical Network

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jadebutterfly872
29 Jun 2018
School
University of Melbourne
Department
Biology
Course
BIOL10005
Professor
All

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LECTURE 11 - Manipulation of the genetic material
PCR
1. DENATURATION: Heat DNA strands to approx 90-95° to break H-bonds and
separate the DNA strands
2. BIND PRIMERS: Cool to approx 50° to anneal/attach primers (single stranded
DNA) which pair with the “region of interest” (at the 3’ end of DNA strands) initiate
replication
oPrimers indicate the region to be copied
oLarge amounts of unbound of nucleotides are also added
oReduced the temperature to allow H-bonds to form
3. Raise temp to 72°: Taq/DNA polymerase copies strands (i.e. by adding
nucleotides onto the primer to form new complementary strands of DNA)
DNA grows in 5’à3’ direction: nucleotides are added to the 3’ end of each strand
1. Cycle is repeated until enough DNA is formed
GEL ELECTROPHORESIS
DNA standard of known size
Separation technique that separates DNA fragments according to their size/charge
using an electric circuit
CLONING AND MANIPULATION OF DNA
Why?
oLarge scale production of specific protein products
HOW?
oCloning into plasmid
oPlasmid: naturally occurring in bacteria, circle DNA, self-replicating
Enzymes used:
oRestriction enzyme: cut DNA molecules at specific sequence
Sticky ends
oDNA ligase: join DNA fragments together during DNA replication
Also join diff DNA fragments with complementary ends together
Transformation:
oBacteria w/ new DNA = transformed
oNot all will have taken up the plasmid
oChecking to see whether bacteria has transformed: blue-white screening or
gel electrophoresis
Cloning DNA:
Allows us to examine gene function as well
oDelete gene
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Document Summary

Denaturation: heat dna strands to approx 90-95 to break h-bonds and separate the dna strands. Bind primers: cool to approx 50 to anneal/attach primers (single stranded. Dna) which pair with the region of interest (at the 3" end of dna strands) initiate replication: primers indicate the region to be copied o. Large amounts of unbound of nucleotides are also added: reduced the temperature to allow h-bonds to form. Raise temp to 72 : taq/dna polymerase copies strands (i. e. by adding nucleotides onto the primer to form new complementary strands of dna) Dna grows in 5" 3" direction: nucleotides are added to the 3" end of each strand. Cycle is repeated until enough dna is formed. Separation technique that separates dna fragments according to their size/charge using an electric circuit. How: cloning into plasmid, plasmid: naturally occurring in bacteria, circle dna, self-replicating. Enzymes used: restriction enzyme: cut dna molecules at specific sequence.

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Related Questions

Please answer all

Why does a template-dependent DNA polymerase require a primer to initiate DNA synthesis?

DNA polymerase require 5' phosphate to add a new nucleotide

DNA polymerase require 3' hydroxyl group to add a new nucleotide

DNA polymerase require energy by hydrolizing the primers

Which of the following enzymes is template independent?

Reverse transcriptase

Klenow fragment

DNA polymerase

Terminal deoxynucleotidyl transferase

RNA polymerase

The natural function of a DNA ligase enzyme is:

Joining together DNA sequences with compatible cohesive ends from two different organisms to yield a recombinant DNA molecule

Sealing single stranded nicks in double stranded DNA molecules

Joining together two blunt ended fragments of DNA within a cell

Joining together two single stranded DNA molecules

The natural function of 3' -> 5' exonuclease activity of a DNA polymerase is to:

Remove the 5' end of the DNA strand that is being copied

Remove damaged nucleotide from the template strand

Remove nucleotides from the end of DNA to generate blunt ends

Remove incorrect nucleotides from the newly synthesized DNA

Which technique is used to resolve the different sizes of DNA fragments?

Gene cloning

PCR

DNA sequencing

Gel electrophoresis

Which of the following vectors does not contain bacterial origin of replication (oriC)?

plasmid

cosmid

bacterial artificial chromosome

lambda vector

Which process of bacteriophage is not used for gene cloning?

Concatemer formation

Lytic cycle

Lysogenic cycle

viral packaging

What vector would be best suited for creating a contig of bovine (cattle) chromosome 10?

lambda vector

Cosmid vector

P1 vector

Plasmid

E. coli takes up plasmid DNA by which of the following methods?

Transduction

Transformation

Conjugation

Transfection

The following times and temperatures are an example of the steps for PCR. You can use the Figure to help you answer the following questions.

Why is the first step is carried out at 94°C?

To degrade the template

To denature the template

To activate the polymerase

To facilitate the primer binding

What happens in the reaction when the temperature shifts to 55°C during cycling?

the DNA polymerase will carry out DNA synthesis by extending the annealed primers

the DNA polymerase will finish DNA synthesis

the primers anneal to the single-stranded regions of the DNA

the primers anneal to the double-stranded regions of the DNA

During cycling, what occurs when the temperature is at 72°C?

the primers anneal to the double-stranded regions of the DNA

the DNA polymerase will carry out DNA synthesis by extending the annealed primers

the primers anneal to the single-stranded regions of the DNA

the DNA polymerase will finish DNA synthesis

You designed a set of primers to amplify 1 kb DNA with polymerase chain reaction. By which cycle of polymerase chain reaction, we would expect to see the first double stranded DNA with expected size?

The end of cycle 1

The end of cycle 2

The end of cycle3

The end of cycle 4

Double check to see if I got these right? 3 questions

Some Processes Involved in DNA Replication

1.The replication origin is identified.

2 DNA primase builds RNA primer.

3Okazaki fragments are spliced by DNA ligase.

4Helicases bind and uncoil the DNA double helix.

5 RNA primer is the starting point for DNA polymerase.

6 DNA replication ends at the telomere where DNA codes fortermination.

7 DNA polymerase adds complementary nucleotides in the 5' to 3'direction in short segments called Okazaki fragments.

The sequence in which the events numbered above occur during DNAreplication of the lagging strand: Number 1 - 7 in order.

I put: 1,4,2,5,7,3,6

__________________Question 2:

Some Functions of Enzymes

  1. Build DNA strands
  2. Build RNA primers
  3. Build mRNA strand
  4. Cleave DNA strands
  5. Splice Okazaki fragments

Enzyme:
Helicase :4
DNA ligase :5
RNA primase :1
RNA polymerase :3
DNA polymerase:2

Question 3:

like mRNA, tRNA has a ribose sugar, U instead of T, and issingle stranded. Unlike mRNA, which remains a long single strand ofnucleotides, tRNA folds so that some areas pair up.

The resulting structure has an anticodon on one end and a site foran amino acid to attach on the other end. There is basecomplementarity (A pairs with U and G pairs with C) between an mRNAcodon and tRNA anticodon.

If the amino acid tyrosine attaches to a tRNA, which of thefollowing anticodons could be at the opposite end of the tRNAmolecule?

Select one:

a. ATA and ATG

b. UAU and UAC

c. AUA and AUG

d. AGA and AGU

I picked B.

Which of the following did Erwin Chargaff observe?

A)

Base composition changes as the organism ages.

B)

Base composition of DNA does not vary from one species toanother.

C)

In all species the number of adenines equals the number ofguanines.

D)

In all species A + T = G + C.

E)

DNAs isolated from different tissues of the same species havethe same base composition.

The melting temperature (Tm) of a DNA duplex is:

A)

the temperature at which double-stranded nucleic acids degradeinto free nucleotides.

B)

the temperature at which double-stranded DNA is toosingle-stranded to ever renature back to double strands.

C)

the temperature at which double-stranded DNA is completelymelted.

D)

the temperature at which double-stranded DNA has become 50%single-stranded.

E)

the temperature at which double-stranded DNA begins to melt.

Given your knowledge and the descriptions of each of thesemethods, which of the following does NOT rely on the ability ofnucleic acids to hybridize with each other?

A)

Southern blotting for the detection of specific DNA fragmentsthat have been separated by gel electrophoresis.

B)

Colony blot hybridization to identify a specific sequence from acollection of sequences that have been inserted into bacteria.

C)

Northern blotting for the detection of specific RNA fragmentsthat have been separated by gel electrophoresis.

D)

A DNA oligonucleotide microarray incubated with probes made fromcDNA.

E)

Western blotting for the detection of specific proteins thathave been separated by gel electrophoresis.

Which of the following is a possible reason why DNA uses thymineinstead of uracil?

A)

Cytidine is deaminated to uracil so it would be difficult forany repair mechanism to differentiate between uracil that belongsin the sequence and uracil that resulted from deamination.

B)

Adenine is deaminated to uracil so it would be difficult for anyrepair mechanism to differentiate between uracil that belongs inthe sequence and uracil that resulted from deamination.

C)

When uracil is bound to a deoxyribose it will become morereactive, forming covalent bonds with other bases.

D)

None of the above.

Which of the following is an enzyme used in cloning to breakcovalent bonds?

A)

DNA ligase

B)

Restriction endonuclease

C)

Reverse transcriptase

D)

DNA polymerase I

E)

Alkaline phosphatase

Which of the following could you use to remove the 5' phosphatesafter cleavage of a plasmid with a single type of restrictionenzyme to ensure that the plasmid would not simply fuse backtogether upon addition of ligase?

A)

Reverse transcriptase

B)

Alkaline phosphatase

C)

DNA ligase

D)

DNA polymerase I

E)

Restriction endonuclease

Which of the following would you use to create a cDNA librarythat you would not use for a genomic library?

A)

Alkaline phosphatase

B)

Reverse transcriptase

C)

Restriction endonuclease

D)

DNA ligase

E)

DNA polymerase I

A plasmid vector typically has which of the followingfeatures?

A)

An origin of replication

B)

A selectable marker to ensure that the only bacteria growingcontain the intact or recombinant vector.

C)

Unique restriction sites for insertion of DNA.

D)

Small size to facilitate entry into the cell and biochemicalmanipulation of the DNA.

E)

All of the above are features of plasmid vectors.

Which of the following is FALSE of genomic or cDNAlibraries?

A)

Genes represented in cDNA libraries include control sequencesthat direct transcription.

B)

cDNA libraries contain cDNAs made from mRNA.

C)

Genomic libraries contain fragments of genomic DNA that aregenerated by partial digests with restriction enzymes.

D)

Genomic libraries usually contain large fragments and are likelyto be constructed in YAC or BAC vectors.

E)

cDNA libraries contain sequences from expressed genes only.

Which of the following is NOT a typical component of apolymerase chain reaction (PCR)?

A)

dNTPs

B)

DNA containing the sequences to be amplified

C)

DNA ligase to connect the fragments together

D)

Primers complementary to each end of the sequence to beamplified

E)

Heat stable Taq polymerase

How can a fusion protein be formed?

A)

Site-directed mutagenesis where a fragment is replaced with asynthetic sequence containing the altered DNA sequence.

B)

Two proteins can recombine in the bacterial cell to form a newhybrid protein.

C)

Portions of two different genes are ligated together to create anew hybrid gene. When expressed the gene product is a fusion of thetwo gene products.

D)

Using oligonucleotide-directed mutagenesis to introduce specificmutations that cause the expressed protein to fuse to otherproteins.

E)

A portion of the gene can be removed to form a shorter gene andtherefore a shorter protein.

Why is green fluorescent protein (GFP) so useful in visualizingfusion proteins in eukaryotes?

A)

The GFP protein recognizes and binds to fusion proteins allowingthe location of the fusion protein to be determined.

B)

The reaction is only dependent on the presence of GFP and oneother cofactor.

C)

It is easily cloned and expressed in bacterial cells.

D)

The presence of just a few molecules of GFP fusion proteins canbe sufficient for observing the proteins microscopically allowingthe study of its location and movements in the cell.

E)

It is derived from fire flies, which are easy to cultivate inthe lab.

You wish to determine the cellular location of a protein butunfortunately it is not particularly antigenic (can t develop astrong antibody response). How could you solve this problem?

A)

Create a fusion protein with an epitope tag that can bevisualized by incubation with fluorescently tagged antibody to theepitope.

B)

Create a fusion with green fluorescent protein (GFP).

C)

Express the protein as a fusion with biotin. The addition of GFPwill cause fluorescence.

D)

A and B might work.

E)

A, B, and C might work.

F)

None of the above will work.

A positive result for the yeast two-hybrid analysis means thefollowing:

A)

The proteins that you are studying can be made into fusionproteins, but do NOT interact with each other within the nucleus ofyeast.

B)

A fusion protein containing the Gal4p activation domain, hasbound to RNA polymerase resulting increase transcription ofnumerous genes, including the reporter gene, and formation ofcolonies.

C)

Two fusion proteins, one containing the Gal4p binding sitedomain and one containing the Gal4p activation domain, haveinteracted through their Gal4p domains resulting in transcriptionof the reporter gene.

D)

Two fusion proteins interact with each other and because of theGal4p binding site domain and the Gal4p activation domain containedin each fusion protein, the reporter genes are activated and thecells die resulting in no colonies.

E)

The colonies growing are expressing the two fusion proteins, butthe two proteins have not interacted with each other.

Which of the following is the approximate size of the humangenome?

A)

3 Gm

B)

300 Kb

C)

3 Mb

D)

3 Gb

E)

3 Mm

Which of the following is correct about the structure orlocation of genes?

Question 22 options:

A)

Prokaryotic genes typically possess their own regulatorysequences that only control expression of a single gene.

B)

Eukaryotic genes often encode introns that are spliced outbefore translation.

C)

Eukaryotic genes are mostly located at the telomeres and thecentromeres of chromosomes.

D)

Eukaryotic genes are typically arranged in operons.

E)

Prokaryotic genes often encode exons that are spliced out beforetranslation