DNA cloning: experimental gene expression introduction to genomes

Question 1

What are the steps of recombinant DNA technology? (4)

Answer 1

-Vector+ DNA fragment
-Recombinant DNA
-Replication of recombinant DNA within host cells
-Isolation, sequencing, and manipulation of purified DNA fragment

Question 2

A vector is a fragment of DNA which includes a: (3)

Answer 2

-replication origin
-restriction enzyme
-some selectable marker

Question 3

What are vectors?

Answer 3

pieces of DNA that have the ability to replicate in a host cell.

Question 4

What is a plasmid? (6)

Answer 4

-most common vector used in recombinant DNA technologies
-circular
-double-stranded DNA (dsDNA)
-extrachromosomal
-found in bacteria and lower eukaryotes
-replication occurs before cell division

Question 5

What is it called when the restriction site is symmetric?

Answer 5

palindrome

Question 6

What is the role of restriction enzymes?

Answer 6

recognize a particular DNA sequence, bind to it, cleave, or cut the phosphodiester bonds

Question 6

What is the most common restriction enzyme?

Answer 6

ECO-R1

Question 7

What are sticky ends?

Answer 7

When we mix the DNA sample with the chosen restriction enzyme, after digestion, you will have DNA fragments with single-stranded overhangs at the ends. The overhangs are complementary to each other, which makes them “sticky” and able to base-pair with other DNA fragments with complementary overhangs.

Question 8

The vector is only going to pair with….

Answer 8

the genomic DNA that was cut with a compatible restriction enzyme, because the other fragments won’t have the same sticky ends that can anneal with the vector.

Question 9

What is the role of T4 DNA ligase?

Answer 9

T4 DNA ligase catalyzes the formation of covalent bonds between DNA fragments with complementary sticky ends, enabling the creation of recombinant DNA molecules.

Question 10

What is the difference between a single restriction enzyme vs multiple enzymes close together?

Answer 10

Using a single enzyme typically results in a defined direction of DNA insertion, as the enzyme recognizes a specific sequence and cleaves the DNA at that site. In contrast, using multiple enzymes can allow for more flexibility in controlling the direction of insertion, as different enzymes can be chosen to target specific sites in the desired orientation.

Question 11

how do we enzimatically insert DNA into plasmid vector ?

Answer 11

The process involves mixing the DNA fragment and the plasmid vector, both cut with compatible restriction enzymes, and using DNA ligase to seal the fragments together, creating a recombinant plasmid

Question 12

What is a important step to do during transformation regarding the bacteria?

Answer 12

Increasing bacterial permeability to facilitate the introduction of DNA

Question 13

Give examples on ways to increase bacterial permeability to facilitate the introduction of DNA ?

Answer 13

-Some bacteria, ex. E.Coli, can be made more permeable to DNA by treating them with calcium ions (CaCl2)
-Heat pulse: temporary heat stress creates small openings in the bacterial cell membrane, allowing DNA to enter
-Culture on nutrient agar plates containing ampicillin: cells that do not take up plasmid die on ampicillin plates

Question 14

How does plasmid replication happen?

Answer 14

Transformed cells survive and replicate because they incorporate essential genetic elements, such as plasmids or DNA fragments, into their genome, allowing them to resist selection pressures

Question 15

How does cell replication happen after plasmid replication?

Answer 15

After plasmid replication, during cell division, the unequal distribution of plasmids can result in varying copy numbers among daughter cells. This variation occurs due to random segregation and cellular processes, and it can persist through subsequent divisions, affecting the plasmid content of individual cells.

Question 16

What is the differences between genomic libraries and cDNA libraries?

Answer 16

genomic libraries encompass an organism’s entire genomic DNA, including both coding and non-coding regions, while cDNA libraries selectively represent expressed genes by containing complementary DNA copies of mRNA molecules.

Question 17

What are BamHI and Sau3A?

Answer 17

Two different restriction enzymes that are employed for DNA fragmentation

Question 18

Explain both BamHI and Sau3A methods.

Answer 18

In both methods, genomic DNA from the organism of interest is fragmented. With BamHI, the DNA is cut at specific sequences, producing fragments with complementary “sticky ends.” Sau3A, on the other hand, generates blunt-ended fragments.

These DNA fragments are then ligated into a cloning vector, such as a plasmid. In the case of BamHI, the vector also has complementary sticky ends. In contrast, for Sau3A, both the fragments and the vector have blunt ends.

After ligation, the recombinant DNA molecules are introduced into host cells through transformation. Bacterial cells are often used as hosts.

To identify successful transformations, cells are grown on selective media, which allows only those with the recombinant DNA to survive. Each colony represents a different genomic DNA fragment from the original organism, creating a DNA library.

Question 19

Explain the production of a DNA library while explaining the reverse transcriptase, cloning, etc

Answer 19

To create a cDNA library, begin by isolating mRNA from cells or tissue samples. Then, use reverse transcriptase to convert this mRNA into complementary DNA (cDNA). These cDNA molecules represent expressed genes and form the basis of the library. Next, clone the cDNA into a vector, such as a plasmid, and introduce the recombinant vectors into host bacterial cells through transformation. Select for transformed cells on agar plates with a selective marker. The bacterial colonies that grow represent your cDNA library, containing cDNA copies of mRNA. This method allows for the study of gene expression and the isolation of specific genes for further analysis.

Question 20

what uses can you make of your recombinant DNA
construct?

Answer 20

-Microarray and in situ hybridization techniques reveal
mRNA expression, co-regulation, and localization.
-Recombinant DNA expression vectors enable regulated
expression of exogenous genes and production of
proteins in prokaryotic and eukaryotic cells.

Question 21

What is in situ hybridization?

Answer 21

In situ hybridization is a molecular technique that uses labeled probes to locate and visualize specific DNA or RNA sequences within cells or tissues, helping researchers study gene expression patterns and spatial distribution.

Question 22

What is microarray analysis simultaneously that measures levels of many mRNAs?

Answer 22

Microarray analysis is a technique that simultaneously measures the levels of many mRNAs (messenger RNAs) in a biological sample. In this method, mRNAs are isolated and converted into labeled cDNA using fluorescent dyes or radioactive markers. These labeled cDNA molecules are then hybridized to a microarray chip containing DNA sequences from thousands of genes. The chip is scanned to detect the fluorescence from the labeled cDNA, allowing researchers to quantify gene expression levels. This high-throughput approach provides insights into gene expression patterns and helps researchers understand how genes respond to different conditions or treatments.

Question 23

What is cluster analysis can identify coordinately regulated genes?

Answer 23

Cluster analysis is a computational method used to identify groups of genes that exhibit similar expression patterns. It helps researchers find genes that are coordinately regulated and potentially involved in the same biological processes or pathways.

Question 24

Can eukaryotic proteins be produced in E. coli?

Answer 24

Yes, often with the inducible lac promoter.

Question 25

Where/how can cloned genes be expressed?

Answer 25

Transiently or stably in cultured animal cells.

Question 26

Can retroviral vectors can be used to integrate cloned genes into mammalian genomes?

Answer 26

yes by transient transfection

Question 27

Explain transient vs stable transfection differences.

Answer 27

Main differences:
Transient: Vector has got an origin of replication, able to replicate the plasma (but plasma doesn’t get integrated into the chromosomes and there’s no selection for it- eventually gets lost.

Stable: there is a selectable marker on the vector so that one can continuously select for the cells that have the clone gene introduced- the plasma gets integrated into the host chromosomes and therefore it’s maintained indefinite.