Question

A plasmid used as a cloning vector in E. coli must have…

Does sequence similarity between genes play an important role in assigning gene function?
Successful insertion of a DNA fragment into the multi-cloning region (restriction sites) of a recombinant plasmid is detected by what changes?

Understand the concept of (restriction enzyme produced) DNA fragment separation by gel electrophoresis.

In addition to restriction enzymes, which enzyme(s) are required to insert a fragment of DNA into a cloning vector?

What is complementary DNA (cDNA) produced from (what is the substrate for its synthesis)?

Understand Southern blotting assay in general. What can it be used for?

Understand the processes of transformation, transfection, conjugation and transduction.

Understand the different types of recognition sequences for restriction enzymes (not the actual sequences).

Why has the Polymerase Chain Reaction revolutionized genetics? What does it do?

In gel electrophoresis, which DNA fragment (in terms of size) would migrate further from the sample well?

Understand gene knockout technology (in general, not the specific players).

Can knockout mice serve as model organisms for study of human disease?

Can insulin be created by recombinant DNA technology?

What is needed for a PCR reaction?

Understand applications for Genetic testing (who are candidates to be tested with this technique).

What is a cDNA library?

Answer 1

1. A plasmid should possess the following

(1) an origin of replication, (2) a drug-resistance gene, and (3) a region where DNA can be inserted without interfering with plasmid replication or expression of the drug-resistance gene.

2. YES! sequence similarity between genes play an important role in assigning gene function that is homologous functions. The plasmid is inserted in bacteria and bacteria is cultured to check the presence of newly inserted DNA fragment.

3. Understand the concept of (restriction enzyme produced) DNA fragment separation by gel electrophoresis.

4. Dna ligase will be necessary to rejoin the cut out DNA

5.cDNA is known to be synthesized, or manufactured from an mRNA or messenger RNA template. It is synthesized in a reaction that is catalyzed by the reverse transcriptase and DNA polymerase enzymes.

6. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. For example, Southern Blotting could be used to locate a particular gene within an entire genome. The amount of DNA needed for this technique is dependent on the size and specific activity of the probe

7.

• In transformation, a bacterium takes up a piece of DNA floating in its environment.

• In transduction, DNA is accidentally moved from one bacterium to another by a virus.

• In conjugation, DNA is transferred between bacteria through a tube between cells.

• Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cell

8. four types of restriction enzymes are recognized, designated I, II, III, and IV, which differ primarily in structure, cleavage site, specificity, and cofactors. Types I and III enzymes are similar in that both restriction and methylase activities are carried out by one large enzyme complex, in contrast to the type II system, in which the restriction enzyme is independent of its methylase. Type II restriction enzymes also differ from types I and III in that they cleave DNA at specific sites within the recognition site; the others cleave DNA randomly, sometimes hundreds of bases from the recognition sequence. Several thousand type II restriction enzymes have been identified from a variety of bacterial species. These enzymes recognize a few hundred distinct sequences, generally four to eight bases in length. Type IV restriction enzymes cleave only methylated DNA and show weak sequence specificity.

9. The polymerase chain reaction (PCR) revolutionized the field of molecular biology. From extremely tiny amounts of DNA, regions can be amplified into millions of copies for further analysis

10.  Smaller molecules migrate through the gel more quickly and therefore travel further than larger fragments that migrate more slowly and therefore will travel a shorter distance.

11. A gene knockout is a genetic technique in which one of an organism's genes is made inoperative. However, it can also refer to the gene that is knocked out or the organism that carries the gene knockout. Knockout organisms or simply knockouts are used to study gene function, usually by investigating the effect of gene loss.

12. Yes because mice genome is very similar to human genome.

13. Recombinant DNA is a technology scientists developed that made it possible to insert a human gene into the genetic material of a common bacterium. This “recombinant” micro-organism could now produce the protein encoded by the human gene. THat is how Scientists harvest the insulin from the bacteria

14. For PCR there are five chemical componentsneeded, including a DNA template, DNA polymerase enzyme, primers, nucleotides and reaction buffer.

15. Genetic testing can provide information about a person's genes and chromosomes. tthe applications are

-Newborn screening is used just after birth to identify genetic disorders that can be treated early in life.

-Diagnostic testing is used to identify or rule out a specific genetic or chromosomal condition.

-Carrier testing is used to identify people who carry one copy of Forensic testing uses DNA sequences to identify an individual for legal purposes.a gene mutation that, when present in two copies, causes a genetic disorder.

-Prenatal testing is used to detect changes in a fetus's genes or chromosomes before birth.

-Preimplantation testing, also called preimplantation genetic diagnosis (PGD), is a specialized technique that can reduce the risk of having a child with a particular genetic or chromosomal disorder.

-Predictive and presymptomatic types of testing are used to detect gene mutations associated with disorders that appear after birth, often later in life

-Forensic testing uses DNA sequences to identify an individual for legal purposes.

16. cDNA library, mRNA is taken from specific cells of an organism, and then cDNA is made from that mRNA in a reaction which is catalyzed by an enzyme.