Analysis of human alu sequences by polymerase chain

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By manipulating the molecular machinery of heat-resistant microorganisms which results in the exponential amplification of DNA via PCR, analysis can be carried out to then characterize and compare various Alu sequences of human genomic DNA.

DNA is a substance that contains hereditary material in biological organisms (Clark 2010). To analyze it, it has to be purified from the medium it is found. It can be taken out of cells and separated from the mixture of cell debris via sedimentary techniques (Walker 2008). To make sure that the DNA is intact and not subject to degradation it has to be treated with chemicals that prevent DNA degrading enzymes from cleaving the DNA (Walker 2008).

PCR is fast and useful but the technique used requires a step of measuring the size of the sequences using agarose gel electrophoresis. Polymerase Chain Reaction (PCR) is a DNA amplification technique which can yield synthesized DNA fragments of a desired size and specificity (van Pelt-Verkuil 2008). Overall it is a process that requires the denaturation, hybridization and extension of DNA by manipulations of molecular machinery and temperature (van Pelt-Verkuil 2008). A user designs primers, which are short segments of DNA, for the heat-resistant polymerases to initiate synthesis, these pairs of upper and lower primers hybridize to the gene of interest (Clark 2010; van Pelt-Verkuil 2008). The great advantage of PCR is that it is specific since the primers are implemented to complement the template strand desired by the user (Clark 2010). The primers attach to the DNA it is complement to, resulting in a double-stranded DNA (dsDNA) strand specific to the sequence of interest (Lui 2009).

The beginning and the end of the sequence's oligonucleotides are required to limit the polymerase's synthesis activity to the sequence of interest (Lui, 2009). A DNA polymerase synthesizes new DNA molecules from a template molecule from 5' to 3' and the new strand-- under the heated conditions of the reaction that dissociates the hydrogen bonds of the dsDNA nucleotides--as well as the original template can have primers and nucleotides attach and contribute to the chain reaction of PCR as templates upon the addition of the primers (Clark 2010). The DNA polymerase used in the PCR process of DNA dissociation has to be heat resistant; the polymerase from bacteria such as Thermus aquaticus or Pyrococcus furiosis are used (BIOL 466, 2010).

The first step of polymerase chain reaction is to separate the strand of template DNA, which is the first template, to about 90° C for about a minute to denature the DNA (Clark 2010). Primers cannot bind the template DNA at 90° C; the next step requires that the temperature is lowered to about 45 to 60° C for the primers to be able to hybridize to complimentary sequences of the template strands (Clark 2010). Following this step, a 70° C temperature is implemented and maintained for around one minute to 2 minutes to allow the polymerase, which is heat -resistant, to synthesize the new strand with the help of the primers which initiated the nucleotides sequence of the new strand. This process is repeated several times, until there are enough partly double-stranded pieces of DNA that can then be melted and become single- stranded templates for more amplification (Clark 2010). This first cycle doesn't provide that much DNA for primers to bind but several steps cycling to give more D|NA templates increases the amount of templates available to primers and oligos for DNA polymerase to synthesize (Walker 2008 p30).

Human Alu sequences are considered mysterious repetitive copies of DNA sequence within the human genome (Batzer Deininger 2004). The sequence can be recognized and cut by a restriction enzyme AluI (Batzer 2004). Though the sequence is around 300 base pairs long, the sites that are recognized by the restriction enzyme have different locations between individuals in the human population, with commonality between people with similar genetic backgrounds (Batzer 2004). The Alu sequences are mobile elements of the human genome, meaning they are inserted throughout the genetic code at different places (Batzer 2004).  These elements are found on various places on the 23 chromosomal pairs which can then present themselves in homozygous or heterozygous patterns (BIOL 466 Jan 10 2011). These data can be realized on an agarose gel through gel electrophoresis (AGE).

Methods

As per BIOL466 Lab Manual 2011 except P1, PV92 PCR primer quantity of 25 L and the loading dye volume was 5 L, respectively.

Results

For this experiment, in order to analyze Alu sequences, the PCR process is used to produce DNA fragments that are specific to regions of the human genome that contain Alu elements on certain chromosomes (Table 2). The specific primers used included PV92, APO, TPA, ACE, FXIIIB, and HSC3N1 (See below).

(BIOL466 Oligos, 2010)

The DNA fragments on the gel corresponding to participant M. McLean produced poorly visible lanes for the amplified products yet lanes P4 and P6 are visible (Figure 1, right).

The DNA fragments of S. Danji on 2% agarose gel (Figure 1, left). The DNA fragments are well resolved and visible for all DNA fragments. Due to the lack of visualization on the gel of participant M. McLean, comparisons of sample population gels will be compared to S. Danji.

The six primers correspond to a single region on one of six chromosomes (BIOL 466 Lecture 2). These primers are labeled P1 through P6 in the lane assignments of the gels. If there is no designation of the primers, the assignment of primers is assigned left to right after the DNA ladder, unless otherwise indicated. Overall, the amplified product of each of the primers selected exhibited different forms of homozygous and heterozygous Alu elements within the pool of participants from Winter 2011 (Figure 3a). S. Danji gel shows the size of the Alu inserts being 300 bp where present through the implementation of Fermentas GeneRulerâ„¢ 100 bp DNA ladder (Figure 2):

Using ethidium bromide lets the bands bound to it have an intense fluorescent colour under UV light (Walker 2008). The higher the percent agarose the better the resolution of the base pairs of DNA (BIOL 466 Jan 10 2011). For higher resolution gels able to detect smaller base pair differences polyacrylamide gels with silver nitrate could be used (Walker 2008 p 140). It is expected that the results of each participant's PCR when resolved up their respective gels will exhibit very high variability between persons through the number of chromosomal inserts or deletions on the Alu sites of interest.

Figure 3a-Alu PCR for participants Winter 2011 Each gel contains primers PV92, APO, TPA, ACE, FXIIIB, and HSC3N1 and exhibits several different polymorphisms in each lane. All of the gels are homozygous for the HSC3N1 Alu segment. Two separate nucleic acids via gel electrophoresis and using ethidium bromide stain, the concentration of agarose in the gel has to be taken into consideration. The resolution of the fragments of DNA is related to the percent of agarose gel. Molecular weight markers at the percentage used increases the resolution of the base pairs in your nucleic acid separation (Figure 2). The buffer are used and an earth gel electrophoresis contains components that prevent deprotonation of nucleic acids, as well as EDTA to chelate cations involved in enzymatic reactions (Lui 2009 p540).

The DNA fragments from the participants in the PCR experiment from Fall 2010 are also considered in this study (Figure 3b). The experiment was carried out under the same conditions as Winter 2011 and the gels contain P1 through P6 with various dimorphisms, each unique. Each gel here is homozygous for the Alu insert HSC3N1.

These next ones are from earlier semesters. The only used five primers instead of six primers.

Table 1a-- Relative frequencies of various Alu sequence inserts for participant S. Danji

 

S.Danji

S.Danji

S.Danji

 

Rel Freq

Rel Freq

Rel Freq

 

Homozygous +/+

Heterozygous -/+

Homozygous -/-

P=

(-/-)

(-/+)

(+/+)

PV92

1

0

0

1

1.000

0.000

0.000

APO

1

0

0

1

1.000

0.000

0.000

TPA

0

1

0

1

0.000

1.000

0.000

ACE

1

0

0

1

1.000

0.000

0.000

FXIIIB

1

0

0

1

1.000

0.000

0.000

HSC3N1

1

0

0

1

1.000

0.000

0.000

Table 1b-- Relative frequencies of various Alu sequence inserts for Winter 2011 participants

 

Winter 2011

Winter 2011

Winter 2011

 

Rel Freq

Rel Freq

Rel Freq

 

Homozygous +/+

Heterozygous -/+

Homozygous -/-

P=

(-/-)

(-/+)

(+/+)

PV92

2

3

2

27

0.074

0.111

0.074

APO

19

3

1

27

0.704

0.111

0.037

TPA

6

7

6

19

0.316

0.368

0.316

ACE

4

2

2

20

0.200

0.100

0.100

FXIIIB

21

1

0

26

0.808

0.038

0.000

HSC3N1

25

0

0

27

0.926

0.000

0.000

Table 1c-- Relative frequencies of various Alu sequence inserts for Fall 2010 participants

 

Fall 2010

Fall 2010

Fall 2010

 

Rel Freq

Rel Freq

Rel Freq

 

Homozygous +/+

Heterozygous -/+

Homozygous -/-

P=

(-/-)

(-/+)

(+/+)

PV92

0

0

0

8

0.000

0.000

0.000

APO

4

2

0

8

0.500

0.250

0.000

TPA

0

5

0

8

0.000

0.625

0.000

ACE

3

0

0

8

0.375

0.000

0.000

FXIIIB

4

0

0

6

0.667

0.000

0.000

HSC3N1

6

0

0

8

0.750

0.000

0.000

Table 1d-- Relative frequencies of various Alu sequence inserts for Earlier Semesters' participants

 

Fall 2009

Fall 2009

Fall 2009

 

Rel Freq

Rel Freq

Rel Freq

 

Homozygous +/+

Heterozygous -/+

Homozygous -/-

P=

(-/-)

(-/+)

(+/+)

PV92

6

3

7

18

0.333

0.167

0.389

APO

13

3

0

18

0.722

0.167

0.000

TPA

4

7

6

17

0.235

0.412

0.353

ACE

14

2

1

18

0.778

0.111

0.056

FXIIIB

9

5

3

18

0.500

0.278

0.167

Table 1d-- Relative frequencies of various Alu sequence inserts for All participants

 

Winter 2011

Fall 2010

Fall 2009

 

Rel Freq

Rel Freq

Rel Freq

 

Homozygous +/+

Heterozygous -/+

Homozygous -/-

P=

(-/-)

(-/+)

(+/+)

PV92

8

6

9

53

0.151

0.113

0.170

APO

36

8

1

53

0.679

0.151

0.019

TPA

10

19

12

44

0.227

0.432

0.273

ACE

21

4

3

46

0.457

0.087

0.065

FXIIIB

34

6

3

50

0.680

0.120

0.060

HSC3N1

31

0

0

35

0.886

0.000

0.000

Table 2-- The Designations of the gel lanes, their respective contents and the chromosome location.

 

 

 

 

Lane

PCR Reactions

Description

Chromosome Number

P1

PV92

Predicted variant

16

P2

APO

Apolipoprotein

11

P3

TPA

Tissue Plasminogen Activator

8

P4

ACE

Angiotensin I Converting Enzyme

17

P5

FXIIIB

Factor 13B

1

P6

HSC3N1

Human Specific C3N1

14

Discussion

The oligonucleotides (Figure 1) are selected to hybridize to the beginning, or upper part of the 5' end of the DNA fragment of interest as well as the ending, or lower part of the 3' end of the DNA fragment of interest (BIOL 466 lecture 2). It was expected that there would not be any matching gels from different participants. This expectation is based on the calculated probability of the occurrence being 1 in 729 for six primers and three choices for zygosity or 1 in 253 for five primers with the same chance of zygosity (BIOL 466, lect.2).

To compare what relative frequency of the sample of participants have heterozygousity for P1 out of all of the participants, which can be done for all the primers that were tested, the frequency of the insertion is calculated based on the total results (P= n) for a given primer within the sample (Tables 1a-1e) . The total results are based on the presence of signal on the gel-a lack of visualization for any gel lane is not considered part of the population. Then, a comparison of those percentages to values obtained from all samples is calculated (Table 1d).

Since nucleic acids have hereditary information that is unique from person to person this can be used to make a distinction between one individual's genome, called DNA typing, and another person's genome (Lui 2009 p10-11).

The lower bands of Hscn1 are from the hybridization of primers to sequence in later cycles. Most participants are homozygous for the insert and considering the significance of Alu inserts as unexcisible portions of DNA inserted millions of years ago in a common human ancestor, this Alu may accurately type the human species (Lui 2009; Batzer 2002).  Furthermore, the homozygosity can also suggest that the parents of the individual with the insert also had homozygosity for the insert on their chromosome. This is based on the hereditary role of genes and their characteristics being based on the characteristics of the parents (Clark, 2008). The sample size is neither random nor large enough to be a representative sample of the human population yet the relative frequencies of the insert provide some details about the significance of the Alu inserts to human history (Xing 2001). On the other hand, not many participants had noticeable bands for P1. This suggests it is an Alu insert that is not as common an insert among all populations as evidenced by its low frequency (Table 1e). Furthermore the frequency of P2 through P5 is varied enough that one might find these the most useful for typing individual DNA (Xing 2001).

The Alu elements have been cited to hold together daughter cells during cell division, which would be advantageous to the successful division of viable cells with viable chromosomes (BIOL 466, lect. 2). Human-specific Alu sequences are sometimes dimorphic are represented in the data in Table 1. The genomic relative frequencies of all the participants in comparison to participant S. Danji show everyone to be homozygous for the insert on Human Specific 3CN1. Human diploidy is responsible for the dimorphism of the gene inserts (Biol 466 Lect. 2 sld 28). Based on the comparison of participant S.Danji's gel to the gels of the other participants and finding not exact match this supports the unlikelihood of the 6 alleles being identical in a given sample of this size (P= 53). The possibility of the "Earlier Semester" gels, with p1 to p5 resolved on the gel, of having the same dimorphism as the more recent gels. This is based on the observation that the entire sample population is homozygous for the HS3CN1 insert. To have all the participants have the Alu insert on both of their respective chromosomes suggests their parent were also homozygous for this insert, suggesting the HS3CN1 Alu insert has been in the human genome since its insertion in an ancestor common to all modern populations. The various zygosity of the other five inserts (p1 - p5) suggest that these inserts may originate from a later ancestor not as common to all of the population and may provide the ethnic and geographic assumptions presented in a more expansive study by Batzer et al.

The addition of the sixth Alu for study increases the unlikelihood of identical gels from 1 in 243 to 1 in 729, or a tripling of the value of running the five inserts. The probability of one person having the same six sequences with the same polymorphism any single participant subject is calculated as 1 in 729. The sample used was 53 human participants of various, undocumented ethnic and genetic backgrounds. It is also notable that there is no identical match found, meaning that no one is related to anyone to significantly as related to the DNA typing characteristic of Alu sequencing. There were similar matches with all 6 having homozygousity for the insert with Winter 2011 #2 and "earlier semesters" #4 which may suggest similar modern ancestry compared to others found in the study.

Finally, the possible sources of error introduced into the gel for M. McLean likely originated from PCR preparations. Specifically the DNA extraction during the saline wash of the mouth may not have been properly executed resulting in inadequate DNA amounts. Further, the PCR mixtures for P1, P2, and P4 were not sufficient enough to produce enough DNA product or DNA bands on the gel. There is also the possibility of error introduced in loading of the gels due to puncturing the wells resulting in the sample seeping out of the gel and into buffer where it cannot be visualized.

Manipulation of molecular machinery of heat-resistant microorganisms such as Taq and/or Pfu and the exponential amplification of DNA, whether genomic or not, can be carried out to then analyze and compare various Alu sequences of human genomic DNA and compare the frequencies of heterozygous and homozygous inserts at certain regions of the chromosomes. The technique is specific and can be applied to forensic analysis, evolutionary studies of human ancestry and possibly gene cloning (Walker 2008).

The population used in this experiment was the Alu sequence population of six different alu sequence site in the human genome. The sample is from participants from the Winter 2011 BIOL 466 semester as well as the Fall 2010 semester. To further extend the sample size, the Winter 2010 and Fall 2009 samples consisting of five of the six sites used in more recent studies (excluding HSNC31) was also included.

There has been research carried out to determine the gene flow and gene frequency within various ethnic populations (Batzer 2002; Nasidze 2001; Xing 2003). This cannot be done here because the sample size is not sufficient data to formally assess these statistics. A random more representative sample will have to be taken where the identities of the participants are not anonymous in order to attain data representative of the whole, as was conducted by Batzer et al (Batzer 2004).

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