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Medical Genetics > 1A- Intro to DNA Technology > Flashcards

1A- Intro to DNA Technology Flashcards

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1
Q

What are some advantages that technology offers with DNA?

A

Cut, modify, reassemble and analyze DNA
Select, characterize, mutate in a predetermined manner and even synthesize individual genes;
Clone and express individual genes to produce the proteins they encode in large amounts (e.g. insulin, human growth factor);
Determine and locate the gene mutations responsible for genetic diseases;
Diagnose sufferers and carriers of genetic diseases;
Plan future therapies that involve manipulation or replacement of genes.

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2
Q

What is a restriction endonuclease?

A

It is an enzyme that cuts DNA at specific nucleotide sequences (restriction sites).

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3
Q

How does a restriction endonuclease cut DNA?

A

To cut the DNA, they need to make 2 incisions, once through each of the sugar-phosphate backbone of the DNA

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4
Q

What are recognition sites?

A

They are locations on DNA that contain specific sequences of nucleotides, which are recognized by restriction enzymes.

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5
Q

In what form does restriction sites usually come in?

A

They are typically palindromic, which means both strand of the DNA have the same base sequence when read in a 5’-3’ direction.

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6
Q

What is a blunt end?

A

the cut DNA is double-stranded at both ends, with no overhangs.

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7
Q

What are sticky ends?

A

the cut products are single-stranded at the ends, with 1 strand overhanging the other.

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8
Q

What is DNA ligase?

A

an enzyme that joins DNA strands together by forming a phosphodiester bond

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9
Q

What is a linker?

A

a piece of DNA that is used to put on a blunt end of a DNA fragment which has been cut by restriction endonucleases.

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10
Q

What is DNA polymerase?

A

It is an enzyme that synthesizes DNA in a 5’-3’ direction from a DNA template

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11
Q

What is reverse transcriptase?

A

Reverse transcriptase is an enzyme that synthesizes DNA from an RNA template.

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12
Q

What type of organisms use reverse transcriptase to make DNA?

A

Viruses

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13
Q

You can use reverse transcriptase in bioengineering on mRNA to produce what?

A

cDNA

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14
Q

What does cDNA not have that regular DNA has?

A

Introns and regulatory regions of the gene. Remember this is coming from mature mRNA, where all the introns are spliced out.

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15
Q

What is hybridization?

A

It is the process of binding 2 complementary strands of nucleic acids together

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16
Q

What is the term of the temperature at which 50% of the DNA is separated?

A

Tm

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17
Q

What is a probe?

A

A probe is a single-stranded DNA or RNA that is used to identify a complementary sequence on a larger single-stranded DNA or RNA

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18
Q

What molecule does western blotting detect?

A

proteins

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19
Q

What does western blotting use as a probe for the proteins?

A

Antibodies

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20
Q

Give the process of a western blot to identify a specific protein

A

proteins added to gel –> gel electrophoresis to separate based on size –> blot to nitrocellulose membrane –> add radioactively-labelled antibodies –> wash –> autoradiography to see binding.

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21
Q

What type of molecule does a Southern blot detect?

A

DNA

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22
Q

What type of probe does a southern blot use to detect DNA?

A

DNA

23
Q

Give the process of a southern blot to find a specific DNA sequence

A

restriction endonucleases cut DNA –> electrophoresis of fragments –> alkaline solution to denature dsDNA –> nitrocellulose membrane to transfer DNA onto it –> bake with UV –> use radioactive DNA probe –> wash –> autoradiographic analysis

24
Q

What type of molecule does Northern blots detect?

A

RNA

25
Q

What type of probe does northern blots use to detect RNA?

A

DNA probe

26
Q

What does an RFLP test for?

A

variations in the lengths of restriction fragments

27
Q

Why would there be variations in lengths of restriction fragments?

A

There could be a mutation in a recognition site so the endonuclease doesnt cut, making the fragment longer and less in #. Or, there could be a mutation in the genome to make a recognition site, making the fragments shorter and more in #.

28
Q

What is a VNTR?

A

VNTR’s are sections of the DNA that contain a repeated tandem # of sequences for a variable # of times

29
Q

What is significant of VNTR’s between different people?

A

They differ in size

30
Q

How can DNA fingerprinting utilize VNTR’s?

A

The restriction fragment patterns produced by the loci can be used to identify individuals.

31
Q

What is chromosome walking?

A

It’s a method used to find, isolate, and clone a particular allele in a gene library

32
Q

What is the process of chromosome walking to find a diseased gene?

A

To locate the particular disease gene, the walking starts at the closest gene that has already been identified, known as a marker gene. Each successive gene in the sequence is tested repeatedly and mapped for their precise location in the sequence. Eventually, walking through the genes reaches the mutant gene in an unmapped sequence

33
Q

What are vectors?

A

A vector is a DNA molecule used as a vehicle to carry foreign genetic material into another cell, where it can be replicated and/or expressed

34
Q

What is bacterial transformation?

A

It is a genetic alteration of a cell resulting from the direct uptake of exogenous DNA from its surroundings

35
Q

What is transfection?

A

Transfection is the process of deliberately introducing nucleic acids into eukaryotic cells, typically from the environment using a ligand as a carrier for endocytotic uptake into the cell

36
Q

Generally,what is cloning?

A

cloning is when you create copies of DNA fragments, cells, or organisms

37
Q

How can you clone a protein to be made?

A

Isolate the segment on DNA where the protein is encoded –> restriction endonuclease to cut portion –> put into plasmid –> transfect into bacteria –> let bacteria grow them suckers –> boom proteins.

38
Q

What is insertional inactivation?

A

It is a technique where a plasmid is used to disable expression of a gene.
The inactivation of a gene is from inserting a fragment of DNA into the middle of its coding sequence.

39
Q

What is a genomic library?

A

A genomic library is a set of host cells that contain all the DNA sequences from the genome of another animal

40
Q

What is a cDNA library?

A

A cDNA library contains all the DNA sequences produced by the reverse transcriptase of mRNA isolated from the cell/tissue of interest

41
Q

What is the difference between a cDNA library and a genomic library? (generally)

A

A cDNA library only has DNA for proteins that are expressed in that type of tissue. A genomic library has all the genes, introns, VNTR’s, and random crap that may not be even used for functional protein synthesis.

42
Q

What is the goal of the PCR?

A

to rapidly produce very large amount of specific segments of DNA.

43
Q

What is the process of the PCR?

A

get DNA –> large amounts of primers added –> add lots of ACTG –> add Taq polymerase –> heat to separate strands –> primers align to DNA sequence you want to amplify –> cool –> primers bind to DNA –> Taq synthesizes off primers in 5’-3’ –> repeats until you stop.

44
Q

How are restriction endonucleases named?

A

Restriction enzymes are named for the bacterium from which they were isolated. They always use the first letter of the genus and then the first 2 letters of the species for the name.
-For example, Escherichia coli RY13’s restriction enzyme is EcoR1.

45
Q

What is recombinant DNA?

A

Recombinant DNA is composed of molecules of DNA from different sources that have been recombined in-vitro

46
Q

How can 2 random DNA molecules get bound together?

A

The sticky ends of 2 unrelated DNA fragments can be joined to each other if they have sticky ends that are complementary

47
Q

How can recombinant DNA be used in bioengineering?

A

When you make a new DNA sequence, you can throw it into a plasmid and incorporate it into bacteria. Therefore, you can start making proteins that would have otherwise not have been synthesized before from those bacteria

48
Q

What types of proteins can recombinant DNA make?

A

insulin, vaccines, HGH, factor VIII, among other proteins for deficiencies in patients.

49
Q

What are siRNA?

A

They are double-stranded RNA molecules that interfere with the expression of specific genes with complementary nucleotide sequences.

50
Q

What is the size of siRNA?

A

They are small, typically 20-25 base pairs long.

51
Q

siRNA are exogenous, synthetic RNA that are trying to be used therapeutically to do what?

A

They are currently being tried to be used to knock out genes in various pathogenic pathways.

52
Q

What are miRNA?

A

They are small non-coding RNA molecules that are found in plants and animals, which functions in transcriptional and post-transcriptional regulation of gene expression

53
Q

What is the function of miRNA?

A

They typically bind with complementary mRNA’s and cause gene silencing. Binding causes the mRNA to be degraded or blocked from translation

Medical Genetics (13 decks)

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