Primer Optimization for DNA Methylation Analysis in 22q11.2 Deletion Target Genes Open Access

Thati, Apoorwa (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/3n203z99k?locale=en
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Abstract

Methylation of cytosine residues of CpG (C-phosphate-G) sites is the most characterized of the epigenetic mechanisms and involves the addition of a methyl group onto the 5' position of a cytosine residue. About 60-90% of CpGs are found methylated throughout the genome, but unmethylated CpG sites can be found clustered in CpG islands most often associated with promoters of the gene. Methylation of these CpG islands is most often associated with transcriptional silencing and has been found to be a significant contributor to gene expression. The mechanism of methylation as a silencing signal is thought to occur by either recruitment of repressive transcriptional silencing machinery or by steric hindrance, preventing the binding of transcriptional factors. We are investigating the methylation modifications of genes CLTCL1 and DGCR8 in the typically deleted region of patients with 22q11 Deletion Syndrome, by designing and optimizing successful primers for bisulfite treated DNA. Future examination of the methylation patterns of these target genes in patients will be done to help clarify the mechanistic connection between 22q11 Deletion Syndrome and schizophrenia.

Table of Contents

Table of Contents

INTRODCUTION 1

MATERIAL AND METHODS 9

DNA Methylation Analysis of Target Genes 9

Obtaining Patient Samples 10

Prioritization of Target Genes 10

Choosing cells to implement protocol 10

Overview of Bisulfite Conversion Sequencing Approach 10

Primer Design for Bisulfite Converted DNA 12

Part I 12

Figure I 12

Figure 2 12

Part II 14

Figure 3 15

Figure 4 15

Table 1 16

Table 2 17

Table 3 18

Optimization of PCR input DNA 19

DNA Extraction 19

Sodium Bisulfite Conversion 19

Polymerase Chain Reaction Optimization 20

Gel Electrophoresis 21

1st Round of PCR Optimization 21

Table 4 21-22

Obtaining control DNA - 100% and 0% 22

2nd Round of PCR Optimization 22

Table 5 22

Table 6 23

Preparation of Patient Samples 23

Table 7 23

3rd Round of PCR 23

Table 8 24

Repeated PCRs 24

PCR Purification 24

4th Round of PCR Optimization - Temperature Gradient 24

Table 9 25

Table 10 25

Table 11 26

Table 12 26

5th round of PCR 27

Table 13 27

Table 14 27

Table 15 27

Table 16 27

Product Purification - Gel or PCR 28

Sequencing 28

RESULTS 30

Prioritization of Target Genes 30

Cell Lines for Optimization 30

Table 17 30

Primer Design 31

Optimization of PCR input DNA 31

Figure 5 32

1st Round of PCR Optimization 32

Figure 6 33-34

2nd Round of PCR 35

Figure 7 36

3rd Round of PCR 37

Figure 8 37-38

Repeated PCRs 39

Figure 9 39

4th Round of PCR Optimization - Temperature Gradient 39

Figure 10 41-42

Table 18 43

5th Round of PCR 43

Figure 11 44-46

DISCUSSION 47

Insight from primer design 47

Implications of the findings 49

Conclusion 53

REFERENCES 51

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