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Genetic innovations- Block 2 > Recombinant dna > Flashcards

Recombinant dna Flashcards

1
Q

Recombinant DNA Technology

A

-It involves combining DNA fragments from different sources, which are not usually found together in nature
-Involves the use of Restriction enzymes along with other strategies for DNA manipulation

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2
Q

Clone

A

Copy of fragment
Copied DNA can be linear or circular

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3
Q

Recombinant DNA

A

Cloned DNA fragment
Recombinant fragment to study its structure

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4
Q

Endonuclease

A

Enzyme that cleaves DNA in the middle of a region/sequence

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5
Q

Exonuclease

A

Enzyme that cleaves at the end of a DNA fragment

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6
Q

Restriction enzymes

A

Endonucleases cleave DNA at specific sites

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7
Q

DNA ligases

A

Enzyme that join two cohesive ends of DNA (ATP dependent)
Eg. T4 DNA ligase

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8
Q

Recombinant DNA technology

A

Used to isolate, replicate, analys genes

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9
Q

Restriction enzymes background

A

-in the 1950s, scientists first began to notice bacteriophages were only able to grow in certain bacterial host strains
-This was called “host-controlled variation” and was explained to occur due to the presence of restriction enzymes

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10
Q

Res- 1970

A

-in the 1970, Werner Weber, Hamilton Smith and Daniel Nathan’s shared the Nobel prize in medicine for discovery of Restriction enzymes
-they discovered that bacteria encoded a restriction factor, called Endonucleases R, that prevented bacteriophages from growing certain hosts while working with Simian Virus
- enzymes selectively cut bacteriophages but not bacterial FNA into fragments of specific and consistent length
-Nathan’s realised it would be a useful tool to map SV40 DNA and its genes
-Not long after, more restriction enzymes were discovered and developed into tools for molecular biologists

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11
Q

RE binding to target DNA

A
  • Work by shape-to-shape matching
    -DNA sequence with a shape that matches a part of the enzyme= recognition site
    -Wraps around the DNA and cleaves both strands of the DNA molecule
    -Each Restriction enzyme binds to unique recognition site: 4-8 bo in length
    -Restriction enzyme sequences are mostly palindromic eg: AGCT, GGATCC
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12
Q

What is a palindrome

A

-Symmetrical sequence that reads the same on both strands in the 5’ to 3’ direction
-2-fold rotational symmetry
-Can form cruciform structures to which proteins such as restriction enzymes can bind

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13
Q

Target DNA structure for RE sites

A

-Both sides of the DNA strand are cleaved at the same position within the restriction site in each DNA strand
-This can leave either blunt ends (double stranded DNA at the end) or sticky ends (cohesive overhangs)

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14
Q

How RE recognition sites work

A
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15
Q

No. Of restriction enzymes discovered

A

-3000 restriction enzymes that recognize over 230 DNA sequences

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16
Q

Type 1 Re

A

Cleaves DNA at random sites far from its recognition sequence

17
Q

Type II

A

Cleaves DNA at defined positions close to or within its recognition sequence

18
Q

Type IIG

A

Cleaves outside its recognition sequence with both REase and MTase enzymatic activities in the same protein

19
Q

Type IIP

A

Cleaves symmetrical targets and cleavage sites

20
Q

Type IIS

A

Recognizes asymmetric sequences

21
Q

Type III

A

cleaves outside its recognition sequence and require two sequences in opposite orientation Ms within the same DNA

22
Q

Type IV

A

Cleaves modified (eg., methylated) DNA

23
Q

How are RS are utilized

A

-Re are used in multiple techniques such as genome mapping by restriction fragment length polymorphisms (RFLPs) and Molecular cloning

24
Q

Restriction Fragment Length Polymorphisms

A

-DNA variation- different sources- polymorphisms
-Sources of variation- single nucleotide polymorphisms (SNPs), variable number tandem repeats (VNTRs), eg. Microsatellites
-restriction enzymes used to differentiate- differences in length of fragments on the gel
-DNA fingerprinting- genome digested and patterned on gel
-Regions probed for lengths- Southern blotting
-PCR-RFLP-single fragment/region isolated then digested

25
Q

RFLP genotyping

A
26
Q

DNA Fingerprinting

A

-SNPs may result in the gain or loss of a restriction site, this changes the DNA fingerprint pattern which is called a restriction fragment length polymorphism (RFLP)
-Variations in micro satellite sequence lengths
-RFLP patterns can therefore be used to differentiate between DNA from different individuals, which is useful in forensics
-RFLPs can also be used to determine if DNA belongs to very closely related individuals and offers a useful tool for paternity testing
-Genomic DNA is treated with a restriction enzyme and resolved on an agarose gel
-Fragments are then proved with radioactive markers and viewed. Modern day probes include fluorescent markers
-the resulting pattern of fragments is called a DNA fingerprint

27
Q

Multi-Locus probes for RFLP

A

-More information obtained when using multiple probes or probes that bind multiple loci
-Homozygote (AA)- one 5kb band
-Heterozygote (Ab)- three bands- 5kb, 2kb and 3kb
-Both alleles present in extracted DNA
-Band intensity will differ when more DNA is present

28
Q

STR/VNTR influence on RFLP

A

-RFLPs can also arise from larger/smaller regions of repetitive DNA
-Different Individuals may have different lengths of repetitive DNA sequences
-Re binding sites on either side of these repeat regions result in different lengths of probed regions

29
Q

PCR RFLP

A

-The polymerase chain reaction (PCR) is a technique used to amplify specific regions of DNA
-PCR can be combined with RFLP analysis in order to look at a specific RFLP marker
-The gene is first amplified by PCR and then digested with the restriction enzyme
-Digest products are resolved on an agarose gel, and the genotypes of each individual may then be interpreted

30
Q

Restriction Mapping

A

-Restriction mapping is a technique used to identify the position of restriction sites on a cloned fragment of DNA
-The DNA clone can either be linear fragment, cloned by PCR or a circular fragment, such as a plasmid, that can be cloned in a bacterial host, such as E.coli

31
Q

Restriction map

A

-Establishes a number of, order of, and distances between restriction enzyme cleavage sites on cloned DNA segments
-Created by cutting DNA with different Re and separating DNA fragments by gel electrophoresis, which separates fragments by size

32
Q

SNPs

A

Single nucleotide polymorphisms

33
Q

VNTR

A

Variable number tandem repeats
-longer than STR
-Also used to identify individuals
- str is less than 25 base pairs while vntr go up to 300 base pairs
-found in promotor region

34
Q

RFLP

A

Restriction fragment length polymorphisms

35
Q

Satellite DNA

A

-Loci in DNA that have repeating sequences
-Includes STRS and VNTRs
-Is detected by restriction digest
-Areas have high degree of polymorphism where number of repeated is specific to the individual

Genetic innovations- Block 2 (11 decks)

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