How they make a scrimillion copies of the DNA Amplification Capillary Electrophereses...
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How they make How they make a scrimillion a scrimillion copies of the copies of the DNA DNA
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Transcript of How they make a scrimillion copies of the DNA Amplification Capillary Electrophereses...
How they make a How they make a scrimillion copies scrimillion copies
of the DNAof the DNA
How they make a How they make a scrimillion copies scrimillion copies
of the DNAof the DNA
•Amplification•Capillary Electrophereses
•Interpretation
Find the areas of interest
D8S1179D8S1179
D21S11D21S11
AmelogeninAmelogenin
D18S51D18S51D3S1358D3S1358
FGAFGA
vWAvWA
D5S818D5S818
D13S317D13S317
D7S820D7S820
D8S1179D8S1179
D21S11D21S11
AmelogeninAmelogenin
D18S51D18S51D3S1358D3S1358
FGAFGA
vWAvWA
D5S818D5S818
D13S317D13S317
D7S820D7S820
Find the areas before and after the areas of interest (primer binding sites)
So we’ve removed the DNA from the
cells, now what?
PCR me,ASAP!
P C R•PCR = Polymerase chain reaction.
•Used to make scrimillions of copies of the loci of interest.
bases = nucleotides
CGACATGTCAT
TACGTAGGCTAACTGCTACA
GT GATAGCAGCG
TCCGATCTAGC
A
GTCT
CAGA
GTCT
CAGA
GTCT
CAGA
GTCT A
GA
GTCT
CAGA
G CTCT
CAGA
GTCT A
GA
GTCT
CAGA
G CTCT
CAGA
GTCT A
GA
GTCT
CAGA
G CTCT
CAGA
So we can just keep splitting it open and adding bases and make millions and
millions of copies.
• This is amplification.
• Technically, this is not a science term either.
• Amplify: Root words Amplus, large + facere, to make*
*Webster’s New World Compact School and Office Dictionary, at 15, (1989)
Holt Science and Technology, Life Science, p. 137
The loci we care about are tagged with a flourescent dye. . .
Getting to the Test Getting to the Test ResultResult
Getting to the Test Getting to the Test ResultResult
What do you do with a billion copies of markers?
•You inject them into the 310 and get a reading.
ABI 310 Genetic Analyzer: Capillary Electrophoresis
Slide courtesy of Simon Ford
DetectorWindow
Slide courtesy of Simon Ford
Okay, now what?• Meet the 310. . .
ABI Prism 310 Genetic Analyzer
Slide courtesy of John Butler
Put on your thinking caps and open your eyes. . .
What we care about:When did it jump, and how
high?• Length aka
number of repeats?
• Quantity aka how much DNA do they have?
What we care about:When did it jump, and how
high?
• Length aka number of repeats. . .
Length• The smaller
DNA pieces pass through the straw (aka capillary) faster than the bigger pieces. They are recorded first.
Picture courtesy of Carll Ladd
Why do we care about this?Because they go
through the capillary according to size, we know what comes through when.
• So we can label them.
D7
AmpFlSTR® Identifiler™ (Applied Biosystems)
AmpFlSTR® Identifiler™ (Applied Biosystems)
AmpFlSTR® Identifiler™ (Applied Biosystems)
AmpFlSTR® Identifiler™ (Applied Biosystems)
AMEL
D3S1358TH01
TPOX
D2S1338
D19S433
FGA
D21S11
D18S51
CSF1PO
D16S539
D7S820
D13S317
D5S818
VWA
D8S1179
See the funny looking ‘ruler’ at the top? That measures the number of base pairs. ‘X’ is about 106 base pairs long. The ‘24’ at D2 is 344 base
pairs long.
AmpFlSTR® Identifiler™ (Applied Biosystems)
Slide courtesy of John Butler
Why do we care about how long these things are?
The length is one of the things that tells us what these things are.
AMEL
D3S1358TH01
TPOX
D2S1338
D19S433
FGA
D21S11
D18S51
CSF1PO
D16S539
D7S820
D13S317
D5S818
VWA
D8S1179
The importance of color. . .
AmpFlSTR® Identifiler™ (Applied Biosystems)
Slide courtesy of John Butler
Quantity of DNA
•The amount of DNA is measured on the right.
WHAT DOES THIS MEAN??
The DNA profile found at the crime scene “Matches” the DNA
profile said to be from the defendant. . .
Let’s compare DNA profiles:
• Stand up! (Humor me, would ya?)• Match is with female• With white skin• And blue eyes• And red hair• Who was raised catholic• By an Irish mother• And has one child
Who’s in the mix?
• It’s time to stand up!! • Match is with female or male• With white skin or olive skin• And blue eyes or brown eyes• And red hair or black hair• Who was raised catholic or free
spirit• By an Irish mother or an Italian
one• And has one daughter or one son
Who’s better
than you?
State of Texas v. Josiah Sutton (1999)
• Woman raped in car by two men
• Identifies Sutton and Adams
• DNA test on vaginal swab, pubic combings, jeans, and semen stain on car seat
Trial testimony•Everyone has a unique DNA pattern
•Sutton’s pattern was found in the vaginal swab, pubic combings, and jeans
•No testimony about semen on car seat (#1 unknown sample)
•Who’s better than you?
•It’s time to ‘Do!’
8,88,98,108,118,128,138,148,158,168,178,188,19
9,99,109,119,129,139,149,159,169,179,189,19
10,1010,1110,1210,1310,1410,1510,1610,1710,1810,19
11,1111,1211,1311,1411,1511,1611,1711,1811,19
12,1212,1312,1412,1512,1612,1712,1812,19
13,1313,1413,1513,1613,1713,1813,19
14,1414,1514,1614,1714,1814,19
15,1515,1615,1715,1815,19
16,1616,1716,1816,19
17,1717,1817,19
18,1818,19 19,19
Defendant 12,13
8,88,98,108,118,128,138,148,158,168,178,188,19
9,99,109,119,129,139,149,159,169,179,189,19
10,1010,1110,1210,1310,1410,1510,1610,1710,1810,19
11,1111,1211,1311,1411,1511,1611,1711,1811,19
12,1212,1312,1412,1512,1612,1712,1812,19
13,1313,1413,1513,1613,1713,1813,19
14,1414,1514,1614,1714,1814,19
15,1515,1615,1715,1815,19
16,1616,1716,1816,19
17,1717,1817,19
18,1818,19 19,19
Defendant 12,13
Alleles Present 10,12,13,14,
15
8,88,98,108,118,128,138,148,158,168,178,188,19
9,99,109,119,129,139,149,159,169,179,189,19
10,1010,1110,1210,1310,1410,1510,1610,1710,1810,19
11,1111,1211,1311,1411,1511,1611,1711,1811,19
12,1212,1312,1412,1512,1612,1712,1812,19
13,1313,1413,1513,1613,1713,1813,19
14,1414,1514,1614,1714,1814,19
15,1515,1615,1715,1815,19
16,1616,1716,1816,19
17,1717,1817,19
18,1818,19 19,19
Defendant 12,13
Alleles Present 10,12,13,14,
15
8,88,98,108,118,128,138,148,158,168,178,188,19
9,99,109,119,129,139,149,159,169,179,189,19
10,1010,1110,1210,1310,1410,1510,1610,1710,1810,19
11,1111,1211,1311,1411,1511,1611,1711,1811,19
12,1212,1312,1412,1512,1612,1712,1812,19
13,1313,1413,1513,1613,1713,1813,19
14,1414,1514,1614,1714,1814,19
15,1515,1615,1715,1815,19
16,1616,1716,1816,19
17,1717,1817,19
18,1818,19 19,19
Defendant 12,13
Alleles Present 10,12,13,14,
15
8,88,98,108,118,128,138,148,158,168,178,188,19
9,99,109,119,129,139,149,159,169,179,189,19
10,1010,1110,1210,1310,1410,1510,1610,1710,1810,19
11,1111,1211,1311,1411,1511,1611,1711,1811,19
12,1212,1312,1412,1512,1612,1712,1812,19
13,1313,1413,1513,1613,1713,1813,19
14,1414,1514,1614,1714,1814,19
15,1515,1615,1715,1815,19
16,1616,1716,1816,19
17,1717,1817,19
18,1818,19 19,19
Defendant 12,13
Alleles Present 10,12,13,14,
15
8,88,98,108,118,128,138,148,158,168,178,188,19
9,99,109,119,129,139,149,159,169,179,189,19
10,1010,1110,1210,1310,1410,1510,1610,1710,1810,19
11,1111,1211,1311,1411,1511,1611,1711,1811,19
12,1212,1312,1412,1512,1612,1712,1812,19
13,1313,1413,1513,1613,1713,1813,19
14,1414,1514,1614,1714,1814,19
15,1515,1615,1715,1815,19
16,1616,1716,1816,19
17,1717,1817,19
18,1818,19 19,19
Defendant 12,13
Alleles Present 10,12,13,14,
15
8,88,98,108,118,128,138,148,158,168,178,188,19
9,99,109,119,129,139,149,159,169,179,189,19
10,1010,1110,1210,1310,1410,1510,1610,1710,1810,19
11,1111,1211,1311,1411,1511,1611,1711,1811,19
12,1212,1312,1412,1512,1612,1712,1812,19
13,1313,1413,1513,1613,1713,1813,19
14,1414,1514,1614,1714,1814,19
15,1515,1615,1715,1815,19
16,1616,1716,1816,19
17,1717,1817,19
18,1818,19 19,19
Defendant 12,13
Alleles Present 10,12,13,14,
15
8,88,98,108,118,128,138,148,158,168,178,188,19
9,99,109,119,129,139,149,159,169,179,189,19
10,1010,1110,1210,1310,1410,1510,1610,1710,1810,19
11,1111,1211,1311,1411,1511,1611,1711,1811,19
12,1212,1312,1412,1512,1612,1712,1812,19
13,1313,1413,1513,1613,1713,1813,19
14,1414,1514,1614,1714,1814,19
15,1515,1615,1715,1815,19
16,1616,1716,1816,19
17,1717,1817,19
18,1818,19 19,19
Defendant 12,13
Alleles Present 10,12,13,14,
15
8,88,98,108,118,128,138,148,158,168,178,188,19
9,99,109,119,129,139,149,159,169,179,189,19
10,1010,1110,1210,1310,1410,1510,1610,1710,1810,19
11,1111,1211,1311,1411,1511,1611,1711,1811,19
12,1212,1312,1412,1512,1612,1712,1812,19
13,1313,1413,1513,1613,1713,1813,19
14,1414,1514,1614,1714,1814,19
15,1515,1615,1715,1815,19
16,1616,1716,1816,19
17,1717,1817,19
18,1818,19 19,19
Defendant 12,13
Alleles Present 10,12,13,14,
15
8,88,98,108,118,128,138,148,158,168,178,188,19
9,99,109,119,129,139,149,159,169,179,189,19
10,1010,1110,1210,1310,1410,1510,1610,1710,1810,19
11,1111,1211,1311,1411,1511,1611,1711,1811,19
12,1212,1312,1412,1512,1612,1712,1812,19
13,1313,1413,1513,1613,1713,1813,19
14,1414,1514,1614,1714,1814,19
15,1515,1615,1715,1815,19
16,1616,1716,1816,19
17,1717,1817,19
18,1818,19 19,19
Defendant 12,13
Alleles Present 10,12,13,14,
15
8,88,98,108,118,128,138,148,158,168,178,188,19
9,99,109,119,129,139,149,159,169,179,189,19
10,1010,1110,1210,1310,1410,1510,1610,1710,1810,19
11,1111,1211,1311,1411,1511,1611,1711,1811,19
12,1212,1312,1412,1512,1612,1712,1812,19
13,1313,1413,1513,1613,1713,1813,19
14,1414,1514,1614,1714,1814,19
15,1515,1615,1715,1815,19
16,1616,1716,1816,19
17,1717,1817,19
18,1818,19 19,19
Defendant 12,13
Alleles Present 10,12,13,14,
15
8,88,98,108,118,128,138,148,158,168,178,188,19
9,99,109,119,129,139,149,159,169,179,189,19
10,1010,1110,1210,1310,1410,1510,1610,1710,1810,19
11,1111,1211,1311,1411,1511,1611,1711,1811,19
12,1212,1312,1412,1512,1612,1712,1812,19
13,1313,1413,1513,1613,1713,1813,19
14,1414,1514,1614,1714,1814,19
15,1515,1615,1715,1815,19
16,1616,1716,1816,19
17,1717,1817,19
18,1818,19 19,19
Defendant 12,13
Alleles Present 10,12,13,14,
15
8,88,98,108,118,128,138,148,158,168,178,188,19
9,99,109,119,129,139,149,159,169,179,189,19
10,1010,1110,1210,1310,1410,1510,1610,1710,1810,19
11,1111,1211,1311,1411,1511,1611,1711,1811,19
12,1212,1312,1412,1512,1612,1712,1812,19
13,1313,1413,1513,1613,1713,1813,19
14,1414,1514,1614,1714,1814,19
15,1515,1615,1715,1815,19
16,1616,1716,1816,19
17,1717,1817,19
18,1818,19 19,19
Defendant 12,13
Alleles Present 10,12,13,14,
15
8,88,98,108,118,128,138,148,158,168,178,188,19
9,99,109,119,129,139,149,159,169,179,189,19
10,1010,1110,1210,1310,1410,1510,1610,1710,1810,19
11,1111,1211,1311,1411,1511,1611,1711,1811,19
12,1212,1312,1412,1512,1612,1712,1812,19
13,1313,1413,1513,1613,1713,1813,19
14,1414,1514,1614,1714,1814,19
15,1515,1615,1715,1815,19
16,1616,1716,1816,19
17,1717,1817,19
18,1818,19 19,19
Defendant 12,13
Alleles Present 10,12,13,14,
15
8,88,98,108,118,128,138,148,158,168,178,188,19
9,99,109,119,129,139,149,159,169,179,189,19
10,1010,1110,1210,1310,1410,1510,1610,1710,1810,19
11,1111,1211,1311,1411,1511,1611,1711,1811,19
12,1212,1312,1412,1512,1612,1712,1812,19
13,1313,1413,1513,1613,1713,1813,19
14,1414,1514,1614,1714,1814,19
15,1515,1615,1715,1815,19
16,1616,1716,1816,19
17,1717,1817,19
18,1818,19 19,19
Defendant 12,13
Alleles Present 10,12,13,14,
15
8,88,98,108,118,128,138,148,158,168,178,188,19
9,99,109,119,129,139,149,159,169,179,189,19
10,1010,1110,1210,1310,1410,1510,1610,1710,1810,19
11,1111,1211,1311,1411,1511,1611,1711,1811,19
12,1212,1312,1412,1512,1612,1712,1812,19
13,1313,1413,1513,1613,1713,1813,19
14,1414,1514,1614,1714,1814,19
15,1515,1615,1715,1815,19
16,1616,1716,1816,19
17,1717,1817,19
18,1818,19 19,19
Defendant 12,13
Alleles Present 10,12,13,14,
15
![Capillary thermostatting in capillary electrophoresis · Capillary thermostatting in capillary electrophoresis ... 75 µm BF 3 Injection: ... 25-µm id BF 5 capillary. Voltage [kV]](https://static.fdocuments.net/doc/165x107/5c176ff509d3f27a578bf33a/capillary-thermostatting-in-capillary-electrophoresis-capillary-thermostatting.jpg)
