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2nd year sem 1: Forensic Genetics > Lecture One : Hybridization and DNA cloning techniques ( not finished) > Flashcards

Lecture One : Hybridization and DNA cloning techniques ( not finished) Flashcards

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1
Q

what’s DNA made of?

A

phosphate group
deoxyribose sugar
phosphate group

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2
Q

How many strands does DNA have?

A

2

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3
Q

how many carbons does pentose sugar have?

A

5

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4
Q

How many hydrogen bonds do each base pairings have?

A

AT - 2

CG- 3

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5
Q

whats chromatin?

A

proteins and DNA that are stacked together

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6
Q

minor groove in DNA is _____.

A

smaller

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7
Q

what are Purine bases?

A

A and G

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8
Q

what are pyrimidine bases?

A

T and C

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9
Q

whats a chromosome?

A

dense packets of DNA by which hereditary information is passed from one generation to the next

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10
Q

whats a autosome?

A

any chromosome other than a sex chromosom

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11
Q

whats a Gene?

A

a sequence of DNA nucleotides on a chromosome

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12
Q

whats a Allele?

A

alternative forms of a gene/marker at a specific genetic location

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13
Q

whats a STR?

A

short tandem repeat

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14
Q

whats a SNIP?

A

single nucleotide polymorphism

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15
Q

How do we select the desired target region?

A

hybridisation

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16
Q

How do you amplify and purify the desired target?

A
  1. DNA cloning

2. Polymerase chain reaction (PCR)

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17
Q

what is hybridisation?

A

two strands of DNA joining together

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18
Q

How can you denature DNA?

A
  1. Heating the DNA

2. Exposure to polar chemicals such as formamide or urea

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19
Q

does denaturing DNA do?

A

breaks it apart into single strand does not damage the DNA

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20
Q

what is Re-forming of the DNA strands known as?

A

nucleic acid annealing or hybridization.

21
Q

what can happen when annealing occures?

A

homoduplexes or heteroduplexes

22
Q

whats a heteroduplexes?

A

uplex where base pairing is not perfect across the full

length of a fragment, or when the DNA from each strand is from two different sources.

23
Q

what can DNA pair with?

A

DNA, RNA and oligonucleotide sequences.

24
Q

whats oligonucleotide?

A

short synthesised, single stranded DNA molecule, ~50 nucleotides long also sometimes referred to as a probe.

25
Q

oligonucleotide is also known as a ____.

A

probe

26
Q

what makes a DNA duplex stable?

A

bases matching between the two molecules.

27
Q

when is stability fo a DNA duplex not affected?

A

short non complimentary regions don’t affect the overall stability

28
Q

what is stringency?

A

conditions which will affect weather two strands anneal

29
Q

what happens when the conditions of stringency are too low ?

A

conditions too low, probe/DNA will anneal to non complimentary regions

30
Q

whats the perfect temperature for annealing?

A

temperature just below the melting temperature (T ) of the DNA or the
oligonucleotide being used.

31
Q

what happens when temperature is above Tm?

A

fully based paired hybrids may be unstable.

32
Q

what happens when temperature below 5°C Tm?

A

mis-paired hybrids may be stable.

33
Q

what alters alter hybridization stringency?

A

temperature and salt concentration

34
Q

what will high salt concentration do to stringency?

A

decrease stringency

35
Q

what will low salt concentration do to stringency?

A

achieve high-hybridization stringency.

36
Q

what are the two categories of Hybridization assays?

A

standard assay, reverse assay

37
Q

whats a standard assay?

A

Labeled probe and unlabeled test sample bound to a solid support.

38
Q

what is a reverse assay?

A

Unlabeled probe bound to a solid support and labeled test sample

39
Q

whats microarray hybridization?

A

Uses the reverse assay method, numerous hybridisation reactions happen at the same time.

40
Q

How is microarray hybridisation done?

A
  1. solid support used to create loads of features on a grid
  2. Each feature/square contains identical unlabeled oligonucleotides (probe)
  3. A test sample containing heterogeneous labelled DNA (or RNA) fragments.
  4. After washing the microarray hybridization signal is read using a laser.
41
Q

what do the results of microarray hybridisation show?

A

strong hybridization signal would indicate a lot of complementary sequences between the test sample and the probe in that feature (square on the grid).

42
Q

can microarray hybridisation be done on RNA?

A

yes

43
Q

what are the 5 Hybridization methods in Forensic Science?

A
  1. SLP/MLP identification of RFLPs using Southern Blot technique
  2. Quantification of DNA and first forensic tests using reverse/dot blot technique.
  3. DNA-DNA hybridization in the polymerase chain reaction.
  4. SNP identification using hybridization
  5. Microarray hybridization for biological fluid identification
44
Q

who developed the southern blott technique?

A

Edwin Southern in 1973

45
Q

what happens at the start of a southern blott technique? step 1

A
  • DNA is digested with restriction endonucleases
  • Fragments separated by electrophoresis
  • Target fragments are identified using labelled probe.
46
Q

what happens to the gel in the southern blott technique? step 2

A

Gel is treated with alkali, this denatures DNA, its then transferred to a nitrocellulose or nylon membrane.

47
Q

what is the membrane soaked in? SBT step 3

A

soaked in a solution containing a labelled probe.

48
Q

what happens to the labelled probe? SBT step 4

A

Labelled probe hybridizes to membrane bound DNA fragments, forming heteroduplexes.

49
Q

what can the label be in SBT?

A

radioactive or fluorescent

2nd year sem 1: Forensic Genetics (8 decks)

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