Manipulating DNA and Gene Cloning

Question 1

What are blunt ends?

Answer 1

A fragment of DNA resulting from the breaking of DNA molecule in which there are no unpaired bases or overhangs in theend, hence, both strands are of the same length.

Question 2

What is cDNA?

Answer 2

complementary DNA: DNA synthesized from a single stranded RNA (e.g., messenger RNA (mRNA) or microRNA) template in a reaction catalyzed by the enzyme reverse transcriptase.cDNAis often used to clone eukaryotic genes in prokaryotes.

Question 3

What is a cDNA Library?

Answer 3

Collection of cloned DNA molecules representing complementary DNA copies of the mRNA produced by a cell

Question 4

What is the Chain Termination Method?

Answer 4

a method of DNAsequencingfirst commercialized by Applied Biosystems, based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.

Question 5

What is Comparative Genomics?

Answer 5

a field of biological research in which thegenomicfeatures of different organisms are compared. Thegenomicfeatures may include the DNA sequence, genes, gene order, regulatory sequences, and othergenomicstructural landmarks.

Question 6

What is DNA cloning?

Answer 6

(1) the act of making many identical copies (typically billions) of a DNA molecule - the amplification of a particular DNA sequence. (2) Also, the isolation of a particular stretch of DNA (often a particular gene) from the rest of the cell’s genome

Question 7

What is a DNA Library?

Answer 7

Collection of cloned DNA molecules, representing either an entire genome (genomic library) or complementary DNA copies of the mRNA produced by a cell (cDNA library)

Question 8

What is Electroporation?

Answer 8

the action or process of introducing DNA or chromosomes into bacteria or other cells using a pulse of electricity to briefly open the pores in the cell membranes.

Question 9

What is gene addtion?

Answer 9

an insertion (also called an insertion mutation) is theadditionof one or more nucleotide base pairs into a DNA sequence. Insertions can be anywhere in size from one base pair incorrectly inserted into a DNA sequence to a section of one chromosome inserted into another.

Question 10

What is gene knockout?

Answer 10

agenetic technique in which one of an organism’sgenesis made inoperative (“knocked out” of the organism)

Question 11

What is gene replacement

Answer 11

a genetic technique in which one of an organisms genes is removed and replaced with another gene

Question 12

What is a genomic library?

Answer 12

Collection of cloned DNA molecules representing an entire genome

Question 13

What is homologous recombination?

Answer 13

Genetic exchange between a pair of identical or very similar DNA sequences, typically those located on two copies of the same chromosome. Also a DNA repair mechanism for double-strand breaks

Question 14

What is the Human Genome Project?

Answer 14

an international scientific researchprojectwith the goal of determining the sequence of nucleotide base pairs that make uphumanDNA, and of identifying and mapping all of the genes of thehuman genomefrom both a physical and a functional standpoint.

Question 15

What does in vitro mean?

Answer 15

performed or taking place in a test tube, culture dish, or elsewhere outside a living organism.

Question 16

What are microarrays?

Answer 16

a grid of DNA segments of known sequence that is used to test and map DNA fragments, antibodies, or proteins.

Question 17

What is a nucleoside triphosphate (NTP)?

Answer 17

amoleculecontaining anucleosidebound to threephosphategroups

Question 18

What is particle bombardment?

Answer 18

a technique which can be used to introduce foreign DNA to a cellculture

Question 19

What is a plasmid vector?

Answer 19

small, circular molecules of double-stranded DNA derived from plasmids that occur naturally in bacterial cells; widely used for gene cloning

Question 20

What is a Poly A tail?

Answer 20

a stretch of RNA that has only adenine bases

Question 21

What is PCR?

Answer 21

Polymerase Chain Reaction: technique for amplifying specific regions of DNA by the use of sequence-specific primers and multiple cycles of DNA synthesis, each cycle being followed by a brief heat treatment to separate complementary strands

Question 22

What are polymorphisms?

Answer 22

describes genome sequences that coexist as two or more sequence variants at high frequency in a population

Question 23

What is primer walking?

Answer 23

asequencingmethod of choice for sequencingDNAfragments between 1.3 and 7kilobases. Such fragments are too long to be sequenced in a single sequence read using thechain termination method. This method works by dividing the long sequence into several consecutive short ones

Question 24

What is recombinant DNA technology?

Answer 24

collection of techniques by which DNA segments from different sources are combined to make a new DNA, often called a recombinant DNA. Recombinant DNAs are widely used in the cloning of genes, in the genetic modification of organisms, and in the production of large amounts of rare proteins

Question 25

What are restriction nucleases?

Answer 25

one of a large number of nucleases that can cleave a DNA molecule at any site where a specific short sequence of nucleotides occurs. Extensively used in recombinant DNA technology

Question 26

What is shotgun sequencing?

Answer 26

Shotgun sequencing involves randomly breaking up DNA sequences into lots of small pieces and then reassembling the sequence by looking for regions of overlap.

Question 27

What is site-directed mutagenesis?

Answer 27

a method to create specific, targeted changes in double stranded plasmid DNA. It is used for investigating the structure and biological activity of DNA, RNA, and protein molecules, and for protein engineering.

Question 28

What is southern blotting?

Answer 28

a procedure for identifying specific sequences of DNA, in which fragments separated on a gel are transferred directly to a second medium on which detection by hybridization may be carried out.

Question 29

What is a sticky end?

Answer 29

an end of a DNA double helix at which a few unpaired nucleotides of one strand extend beyond the other.

Question 30

What is taq polymerase?

Answer 30

an enzyme found in the bacillus Thermus aquaticus, which lives in hot springs; it is heat resistant and thus can endure the high temperatures of the polymerase chain reaction.

Question 31

What are Type II endonucleases?

Answer 31

A type of enzyme that recognizes short, usually palindromic, sequences of 4–8 bp and, in the presence of Mg2+, cleave the DNA within or in close proximity to the recognition sequence.

Question 32

What are Yeast artificial chromosomes?

Answer 32

A vector (carrier) created and used in the laboratory to clone pieces of DNA. A YAC is constructed from the telomeric, centromeric, and replication origin sequences needed for replication in yeast cells.

Question 33

What is one of the simplest ways to clone a section of DNA?

Answer 33

Insert the sequence into the purified DNA genome of a self-replicating genetic element - usually a plasmid vector derived from bacterial cells

Question 34

How is DNA inserted into a plasmid vector?

Answer 34

In order to insert a segment into a plasmid, the circular DNA must first be cut with restriction nuclease to create linear DNA. The DNA to be cloned is then added and the DNA ligase enzyme covalently bonds them together

Question 35

True or False: cDNA clones contain both introns and exons

Answer 35

False - cDNA only contains exons that have been transcribed into mRNA

Question 36

True or False: cDNA libraries are the same for every cell in the one organism

Answer 36

False - different tissues produce distinct sets of mRNA molecules, so distinct cDNA libraries are obtained for each type of cell used

Question 37

How is cDNA synthesised?

Answer 37

Poly-T primer bonds to poly-A tail of mRNA. Reverse transcriptase adds complementary DNA copy of the mRNA. RNase H makes nicks and gaps in the mRNA degrading it. DNA polymerase then synthesises a seocnd cDNA strand making a double-stranded cDNA copy of the original mRNA

Question 38

How is a DNA library made?

Answer 38

Genomic DNA is cleaved into small pieces using restriction endonucleases and then ligated into plasmid vectors, using conditions that favour the insertion of a single DNA fragment into each plasmid molecule. The plasmids are then reintroduced into the bacteria where they replicate

Question 39

Why do bacteria produce restriction endonucleases?

Answer 39

They protect the bacteria from viruses by degrading incoming viral DNA

Question 40

What is a characteristic of a type II endonuclease?

Answer 40

The cleavage site is within or close to the recognition site

Question 41

How long are DNA probes?

Answer 41

Usually around 30 nucleotides long

Question 42

How do bacteria protect themselves from their own endonucleases?

Answer 42

They modify their DNA by adding methyl groups to their adenine or cytosine (depends on bacteria) which blocks the binding of the restriction enzymes

Question 43

How do the polymerase chain reaction (PCR) work?

Answer 43

At the start of the first cycle, the two strands of dsDNA are separated and an appropriate primer binds to each strand. The primers mae the left and right boundaries of the DNA to be amplified. Taq polymerase is then allowed to replicate each strand. This cycle is then repeated as desired

Question 44

What are the major uses of endonucleases?

Answer 44

``` DNA sequencing Gene cloning DNA fingerprinting Gene mapping Gene disruption ```

Question 45

How do you control the direction a gene is added to the DNA?

Answer 45

By using two different restriction endonucleases

Question 46

How is a bacteria with recombinant DNA identified?

Answer 46

Often a gene that gives the bacteria resistance to a certain antibiotic is added along with recombinant gene. That antibiotics will then allow for the selection of bacteria that have successfully incorporated the DNA into their plasmid

Question 47

How does site-directed mutagenesis work?

Answer 47

A plasmid with an inserted gene is separated into single strands where a synthetic oligonucleotude primer containing the desired mutated sequence anneals to an almost complementary site. DNA polymerase completes the second strand. Upon replication of the plasmid, a mutated daughter version will be made using the mutated strand

Question 48

How does particle bombardment work?

Answer 48

Plasmid is coated with gene of interest on tungsten or gold particles. Machine penetrates cell walls and the transgenes make be incorporated into chromosmal DNA. Markers can be used to identify bacteria that have taken up the transgene

Question 49

What do DNA microarrays do?

Answer 49

They analyse gene expression by monitoring mRNA products of thousands of genes at once. The mRNA is converted into cDNA and labelled with a fluorescent probe. The microarray is then scanned to see which genes the probe binds to

Question 50

How does the chain termination method work?

Answer 50

DNA is synthesised in vitro in a mixture that contains ssDNA, DNA polymerase and the four nucleotides. If a dideoxynucleotide anolog of one of the nucleotides is present, it can become incorporated into the growing chain. Because it lacks the 3' OH, the addition of the next nucleotide is impossible so the chain is terminated. This happens at different lengths for each strand so the sequence of the DNA can be determined

Question 51

The study of the relationship within a family of genes sequence homology is called

Answer 51

Comparative genomics