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BMS1062 > Manipulating DNA and Gene Cloning > Flashcards

Manipulating DNA and Gene Cloning Flashcards

1
Q

What are blunt ends?

A

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.

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2
Q

What is cDNA?

A

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.

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3
Q

What is a cDNA Library?

A

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

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4
Q

What is the Chain Termination Method?

A

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.

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5
Q

What is Comparative Genomics?

A

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.

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6
Q

What is DNA cloning?

A

(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

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7
Q

What is a DNA Library?

A

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)

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8
Q

What is Electroporation?

A

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.

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9
Q

What is gene addtion?

A

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.

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10
Q

What is gene knockout?

A

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

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11
Q

What is gene replacement

A

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

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12
Q

What is a genomic library?

A

Collection of cloned DNA molecules representing an entire genome

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13
Q

What is homologous recombination?

A

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

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14
Q

What is the Human Genome Project?

A

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.

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15
Q

What does in vitro mean?

A

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

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16
Q

What are microarrays?

A

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

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17
Q

What is a nucleoside triphosphate (NTP)?

A

amoleculecontaining anucleosidebound to threephosphategroups

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18
Q

What is particle bombardment?

A

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

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19
Q

What is a plasmid vector?

A

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

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20
Q

What is a Poly A tail?

A

a stretch of RNA that has only adenine bases

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21
Q

What is PCR?

A

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

22
Q

What are polymorphisms?

A

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

23
Q

What is primer walking?

A

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

24
Q

What is recombinant DNA technology?

A

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

25
Q

What are restriction nucleases?

A

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

26
Q

What is shotgun sequencing?

A

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

27
Q

What is site-directed mutagenesis?

A

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

28
Q

What is southern blotting?

A

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.

29
Q

What is a sticky end?

A

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

30
Q

What is taq polymerase?

A

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

31
Q

What are Type II endonucleases?

A

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.

32
Q

What are Yeast artificial chromosomes?

A

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 inyeastcells.

33
Q

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

A

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

34
Q

How is DNA inserted into a plasmid vector?

A

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

35
Q

True or False: cDNA clones contain both introns and exons

A

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

36
Q

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

A

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

37
Q

How is cDNA synthesised?

A

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

38
Q

How is a DNA library made?

A

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

39
Q

Why do bacteria produce restriction endonucleases?

A

They protect the bacteria from viruses by degrading incoming viral DNA

40
Q

What is a characteristic of a type II endonuclease?

A

The cleavage site is within or close to the recognition site

41
Q

How long are DNA probes?

A

Usually around 30 nucleotides long

42
Q

How do bacteria protect themselves from their own endonucleases?

A

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

43
Q

How do the polymerase chain reaction (PCR) work?

A

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

44
Q

What are the major uses of endonucleases?

A 
DNA sequencing
Gene cloning
DNA fingerprinting
Gene mapping
Gene disruption
45
Q

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

A

By using two different restriction endonucleases

46
Q

How is a bacteria with recombinant DNA identified?

A

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

47
Q

How does site-directed mutagenesis work?

A

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

48
Q

How does particle bombardment work?

A

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

49
Q

What do DNA microarrays do?

A

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

50
Q

How does the chain termination method work?

A

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

51
Q

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

A

Comparative genomics

BMS1062 (9 decks)

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